Genetic markers for skatole metabolism

ABSTRACT

Novel metabolites and enzymes involved in skatole metabolism are disclosed. The novel metabolites are 3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI); indole-3-carbinol (I-3C); and 2-aminoacetophenone (2-AM). Measuring levels of these metabolites in a pig may be useful in identifying the pig&#39;s ability to metabolize skatole and its susceptibility to boar taint. The novel enzymes involved in skatole metabolism are aldehyde oxidase and CYP2A6. Enhancing the activity of these enzymes may be useful in enhancing skatole metabolism and reducing boar taint. The identification of the enzyme also allows the development of screening assays for substances that interact with these enzymes and skatole metabolism or for genetic screening to identify pigs on the basis of their skatole metabolism. Pigs having high levels of these enzymes may be selected and bred to produce pigs that have a lower incidence of boar taint.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit from U.S. application Ser. No.10/206,118 filed Jul. 29 2002, which is a divisional of U.S. applicationSer. No. 09/672,039, filed Sep. 29,2000 now U.S. Pat. No. 6,448,028which is a continuation of U.S. provisional application No. 60/156,935,filed Sep. 30, 1999 all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel metabolites of skatole and theidentification of novel enzymes involved in the metabolism of skatole.The invention has utility in developing methods to identify and reduceboar taint.

BACKGROUND OF THE INVENTION

Male pigs that are raised for meat production are usually castratedshortly after birth to prevent the development of off-odors and offflavors (boar taint) in the carcass. Boar taint is primarily due to highlevels of either the 16-androstene steroids (especially5α(-androst-16-en-3-one)) or skatole in the fat. Recent results of theEU research program AIR 3-PL94-2482 suggest that skatole contributesmore to boar taint than androstenone (Bonneau, M., 1997).

Skatole is produced by bacteria in the hindgut which degrade tryptophanthat is available from undigested feed or from the turnover of cellslining the gut of the pig (Jensen and Jensen, 1995). Skatole is absorbedfrom the gut and metabolized primarily in the liver (Jensen and Jensen,1995). High levels of skatole can accumulate in the fat, particularly inmale pig, and the presence of a recessive gene Ska.sup.1, which resultsin decreased metabolism and clearance of skatole has been proposed(Lundström et al., 1994; Friis, 1995). Skatole metabolism has beenstudied extensively in ruminants (Smith, et al., 1993), where it can beproduced in large amounts by ruminal bacteria and results in toxiceffects on the lungs (reviewed in Yost, 1989). The metabolic pathwaysinvolving skatole have not been well described in pigs. In particular,the reasons why only some intact male pigs have high concentrations ofskatole in the fat are not clear. Environmental and dietary factors areimportant (Kjeldsen, 1993; Hansen et al., 1995) but do not sufficientlyexplain the reasons for the variation in fat skatole concentrations inpigs. Claus et al. (1994) proposed high fat skatole concentrations are aresult of an increased intestinal skatole production due to the actionof androgens and glucocorticoids. Lundström et al. (1994) reported agenetic influence on the concentrations of skatole in the fat, which maybe due to the genetic control of the enzymatic clearance of skatole. Theliver is the primary site of metabolism of skatole and liver enzymaticactivities could be the controlling factor of skatole deposition in thefat. B.ae butted.k et al. (1995) described several liver metabolites ofskatole found in blood and urine with the major being MII and MIII. MII,which is a sulfate conjugate of 6-hydroxyskatole (pro-MII), was onlyfound in high concentrations in plasma of pigs which were able torapidly clear skatole from the body, whereas high Mm concentrations wererelated to slow clearance of skatole. Thus the capability of synthesisof MII could be a major step in a rapid metabolic clearance of skatoleresulting in low concentrations of skatole in fat and consequently lowlevels of boar taint.

In view of the foregoing, further work is needed to fully understand themetabolism of skatole in pig liver and to identify the key enzymesinvolved. Understanding the biochemical events involved in skatolemetabolism can lead to novel strategies for treating, reducing orpreventing boar taint. In addition, polymorphisms in these candidategenes may be useful as possible markers for low boar taint pigs.

SUMMARY OF THE INVENTION

The present inventors have identified novel metabolites resulting fromthe phase I metabolism of skatole (3-methylindole, 3MI) by porcine livermicrosomes. The metabolites identified are: 3-OH-3-methylindolenine(HMI); 3-methyloxindole (3MOI); indole-3-carbinol (I-3C); and2-aminoacetophenone (2-AM). Measuring levels of these metabolites in apig may be useful in identifying the pig's ability to metabolize skatoleand hence its susceptibility to boar taint.

The present inventors have also determined that one of the metabolitesof skatole, HMI is metabolized to 3-hydroxy-3-methyloxindole (HMOI) byaldehyde oxidase. As a result, enhancing the activity of the aldehydeoxidase may be useful in enhancing skatole metabolism and reducing boartaint. Accordingly, the present invention provides a method forenhancing the metabolism of 3-methylindole and thereby reducing boartaint comprising enhancing the activity of aldehyde oxidase in a pig.The activity of aldehyde oxidase can be enhanced by using substanceswhich (a) increase the activity of aldehyde oxidase; or (b) induce orincrease the expression of the aldehyde oxidase gene.

The present inventors have further determined that the cytochrome P450enzyme, CYP2A6, is also involved in the metabolism of skatole by porcineliver microsomes. As a result, enhancing the activity of the CYP2A6 maybe useful in enhancing skatole metabolism and reducing boar taint.Accordingly, the present invention provides a method for enhancing themetabolism of 3-methylindole and thereby reducing boar taint comprisingenhancing the activity of CYP2A6 in a pig. The activity of CYP2A6 can beenhanced by using substances which (a) increase the activity of CYP2A6;or (b) induce or increase the expression of the CYP2A6 gene.

The identification of enzymes involved in the metabolism of skatoleallows the development of screening assays for substances that interactwith these enzymes in skatole metabolism. The screening assays can beused to identify substances that can be used to reduce or treat boartaint.

The present invention also includes a method for producing pigs thathave a lower incidence of boar taint by selecting pigs that have highlevels of aldehyde oxidase and/or CYP2A6 and breeding the selected pigs.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a chromatographic profile of the main five metabolitesproduced by pig liver microsomes as detected by UV absorption at 250 nm.Retention times correspond as follows: 9.16 min, WV-1; 11.24 min,3-hydroxy-3-methyloxindole; 14.42 min, indole-3-carbinol; 17.51 min,3-methyloxindole; 19.43 min, 2-aminoacetophenone; 22.84 min, parentcompound (3-methylindole). (A) Standard mixture containing 2 .μg/ml ofeach metabolite. (B) Incubation mixture.

FIG. 2 is a UV spectra of (A) UV-1 metabolite [λ_(max) (nm): 204, 238];(B) 3-methyloxindole[λ_(max) (nm): 205, 252]; and (C)3-hydroxy-3-methyloxindole[HMOI: [λ_(max) (nm): 208, 253].

FIG. 3A is an LC-MS spectrum of metabolite UV-1.

FIG. 3B is an MS/MS spectrum of daughter ion of m/z 148.

FIG. 4 is an ¹H-NMR spectrum of metabolite UV-1.

FIG. 5 shows chemical structures and percentages of 3MI metabolitesproduced by pig liver microsomes.

FIG. 6 shows the oxidative conversion of 3-hydroxy-3-methylindolenineinto 3-hydroxy-3-methyloxindole catalyzed by aldehyde oxidase.

FIG. 7 shows the formation of 3-hydroxy-3-methyloxindole (HMOI) from3-hydroxy-3-methylindolenine, catalyzed by porcine cytosol. Each datapoint represents the mean of duplicate assays performed for three pigs.

FIG. 8 shows the menadione-induced inhibition of the formation of3-hydroxy-3-methyloxindole (HMOI) from 3-hydroxy-3-methylindolenine.Each data point represents the mean of duplicate assays performed forthree pigs.

FIG. 9 shows the quinacrine-induced inhibition of the formation of3-hydroxy-3-methyloxindole (HMOI) from 3-hydroxy-3-methylindolenine.Each data point represents the mean of duplicate assays performed forthree pigs.

FIG. 10 shows the plot of back fat 3-methylindole content versus hepaticaldehyde oxidase activity in pigs (n=30). Aldehyde oxidase activitymeasured as nmol of 3-hydroxy-3-methyloxindole (HMOI) formed per mg ofcytosolic protein per min.

FIG. 11 shows the sequence alignment of the CYP2A6 gene (SEQ ID NO:3)and the mutation (SEQ ID NO:1), at nt position 1220, indicated in bold.

FIG. 12 shows the Nucleotides sequence and deduced amino acid sequencesfor the pig cytochrome P450 2A6 cDNA from the liver. CYP2A6 was isolatedfrom a pig cDNA library. (SEQ ID NO:18) The nucleotide sequence has beenregistered in the GenBank (accession number, AY091516). The deducedamino acid sequence is indicated below the corresponding nucleotidesequence. Three active sites for CYP2A6 are underlined. The numbers ofnucleotides and amino acids are indicated in the right.

FIG. 13 shows the alignment of amino acid sequence of human CYP2A6 (SEQID NO:19), CYP2A3 (SEQ ID NO:20) and pig 2A6 (SEQ ID NO:21). Gln104,Phe209 and His477 are reported to be active site for human CYP2A6coumarin 7-hydroxylase activity, oxidative metabolism of nicotine andcotinine. The numbers of amino acids are indicated in the right.Asterisk indicated identical for these active site between human andpig.

FIG. 14 shows the results of Northern blot analysis of the CYP2A6′expression in different porcine tissues. Total RNAs were extracted fromspleen, thymus, liver, lung, muscle, ovary, kidney, small intestineheart and testis, respectively. 20 μg of Total RNA (per lane) wereelectrophoresed on a 1.0% agarose gel containing 2.0 M formaldehyde. TheRNAs were transferred to a nylon membrane and then hybridized withdig-labeled porcine CYP2A6 cDNA.

FIG. 15 shows the Genetic polymorphism, sequencing, western blottinganalysis, and micosomal enzyme activity and skatole level in fat forCYP2A6 in pig liver. A|, PCR-SSCP analysis of CYP2A6 cDNA. M: deletionmutant; W: wild type. B, Sequencing analysis of CYP2A6 for deletionmutant and wild type. M: sequencing data for deletion mutant; W:sequencing data for wild type. C, total proteins from microsome wereseparated in 12% SDS-PAGE electrophoresis, immunoblotted with mouseanti-human monoclonal 2A6-antibody. Duplicated and 40 μg of totalprotein from liver microsome was loaded in each lane. M: total proteinfrom individual that has the deletion mutant for CYP2A6; W: totalprotein from wild type pig liver. D, micosomal CYP2A6 activity andskatole level in fat for both deletion mutant and wild type.

DETAILED DESCRIPTION OF THE INVENTION

I. Skatole Metabolites

The present inventors have identified novel metabolites resulting fromthe phase I metabolism of skatole (3-methyl indole, 3MI) by porcineliver microsomes. The metabolites identified are:3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI);indole-3-carbinol (I-3 C); and 2-aminoacetophenone (2-AM).

Measuring levels of these metabolites in a pig may be useful inidentifying the pig's ability to metabolize skatole and itssusceptibility to boar taint. Accordingly, the present inventionprovides a method of assessing a pig's ability to metabolize 3-methylindole comprising testing a sample from the pig for one or moremetabolites selected from the group consisting of3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI);indole-3-carbinol (I-3C); and 2-aminoacetophenone.

Since skatole metabolites also undergo Phase II sulfation andglucuronidation reactions, the assay may include measuring the sulfationor glucuronidation products of the metabolites. The sample can be anybiological sample from the pig, preferably liver, plasma or fat.Measuring levels of particular metabolites can be used to classify pigsas either good or poor skatole metabolizers. Poor skatole metabolism maybe causative of boar taint and therefore the assay may be useful inidentifying pigs with boar taint or at risk for developing poor taint.Pigs that have a reduced risk for boar taint (i.e., good metabolizers)may be further selected and bred to produce low boar taint pigs.

II. Enzymes

a) Aldehyde Oxidase

The present inventors have determined that one of the metabolites ofskatole, HMI is metabolized to 3-hydroxy-3-methyloxindole (HMOI) byaldehyde oxidase, a cytosolic metalloflavoprotein. The inventors havealso determined that aldehyde oxidase plays an important role in themetabolism of skatole (or 3MI) and that its catalytic activity isrelated to adequate 3MI clearance. As a result, enhancing the activityof the aldehyde oxidase may be useful in enhancing skatole metabolismand reducing boar taint. Accordingly, the present invention provides amethod for enhancing the metabolism of 3-methylindole comprisingenhancing the activity of aldehyde oxidase in a pig. The activity ofaldehyde oxidase can be enhanced by using substances which (a) increasethe activity of aldehyde oxidase; or (b) induce or increase theexpression of the aldehyde oxidase gene. The activity of aldehydeoxidase may also be enhanced using gene therapy whereby a nucleic acidsequence encoding an dehyde oxidase enzyme in introduced into a pigeither ex-vivo or in-vivo. A nucleic acid sequence encoding aldehydeoxidase may be obtained by cloning the pig gene using the informationavailable from the human, bovine and rabbit genes.

As mentioned above, aldehyde oxidase activity is related to 3MIclearance. As a result, testing the enzymatic activity of aldehydeoxidase in a pig can be used to determine a pig's susceptibility to boartaint. Pigs with high aldehyde oxidase activity would be at a lower riskfor boar taint than pigs with a low aldehyde oxidase activity. Pigs withhigh aldehyde oxidase activity may be selected and bred to produce lowboar taint pigs.

Accordingly, the present invention provides a method of determining apig's susceptibility to boar taint comprising determining the activityof aldehyde oxidase in a sample from a pig. Methods for determiningaldehyde oxidase activity are detailed in Example 2.

b) CYP2A6

The present inventors have further determined that the cytochrome P450enzyme, CYP2A6, is also involved in the metabolism of skatole by porcineliver microsomes. As a result, enhancing the activity of CYP2A6 may beuseful in enhancing skatole metabolism and reducing boar taint.Accordingly, the present invention provides a method for enhancing themetabolism of 3-methylindole comprising enhancing the activity of CYP2A6in a pig. The activity of CYP2A6 can be enhanced by using substanceswhich (a) increase the activity of CYP2A6; or (b) induce or increase theexpression of the CYP2A6 gene. The activity of CYP2A6 may also beenhanced using gene therapy whereby a nucleic acid sequence encoding aCYP2A6 enzyme in introduced into a pig either ex-vivo or in-vivo. Anucleic acid sequence encoding CYP2A6 may be obtained by cloning the piggene using the information available from the human gene.

Testing the enzymatic activity of CYP2A6 in a pig can be used todetermine a pig's susceptibility to boar taint. Pigs with high CYP2A6activity would be at a lower risk for boar taint than pigs with a lowCYP2A6 activity. Pigs with high CYP2A6 activity may be selected and bredto produce low boar taint pigs. Accordingly, the present inventionprovides a method of determining a pig's susceptibility to boar taintcomprising determining the activity of CYP2A6 in a sample from a pig.

c) Screening Assays

The identification of enzymes involved in the metabolism of skatoleallows the development of screening assays for substances that interactwith these enzymes and thereby modulate skatole metabolism.

In one aspect, the present invention provides a method of screening fora substance that enhances the activity of aldehyde oxidase or CYP2A6.

In one embodiment of the invention, a method is provided for screeningfor a substance that enhances skatole metabolism in a pig by enhancingaldehyde oxidase activity comprising the steps of:

(a) reacting a substrate of aldehyde oxidase and aldehyde oxidase, inthe presence of a test substance, under conditions such that aldehydeoxidase is capable of converting the substrate into a reaction product;

(b) assaying for reaction product, unreacted substrate or unreactedaldehyde oxidase;

(c) comparing to controls to determine if the test substance selectivelyenhances aldehyde oxidase activity and thereby is capable of enhancingskatole metabolism in a pig. Substrates of aldehyde oxidase which may beused in the method of the invention include HMI which is metabolized toHMOI.

The induction of aldehyde oxidase activity can be measured using avariety of techniques including measuring the levels of the aldehydeoxidase protein or mRNA or by testing for aldehyde oxidase activity.Aldehyde oxidase activity can be measured using various assays includingthe assay described in Example 2 and those described by Rajagopalan etal., 1966.

In another embodiment of the invention, a method is provided forscreening for a substance that enhances skatole metabolism in a pig byenhancing CYP2A6 activity comprising the steps of:

(a) reacting a substrate of CYP2A6 and CYP2A6, in the presence of a testsubstance, under conditions such that CYP2A6 is capable of convertingthe substrate into a reaction product;

(b) assaying for reaction product, unreacted substrate or unreactedCYP2A6;

(c) comparing to controls to determine if the test substance selectivelyenhances CYP2A6 activity and thereby is capable of enhancing skatolemetabolism in a pig.

Substrates of CYP2A6 which may be used in the method of the inventionfor example include skatole and coumarin.

The induction of CYP2A6 activity can be measured using a variety oftechniques including measuring the levels of the CYP2A6 protein or mRNAor by testing for CYP2A6 activity as described in Aitio, 1978.

The aldehyde oxidase and CYP2A6 enzymes used in the method of theinvention may be obtained from natural, recombinant, or commercialsources. Cells or liver microsomes expressing the enzymes may also beused in the method.

Conditions which permit the formation of a reaction product may beselected having regard to factors such as the nature and amounts of thetest substance and the substrate.

The reaction product, unreacted substrate, or unreacted enzyme; may beisolated by conventional isolation techniques, for example, salting out,chromatography, electrophoresis, gel filtration, fractionation,absorption, polyacrylamide gel electrophoresis, agglutination, orcombinations thereof.

To facilitate the assay of the reaction product, unreacted substrate, orunreacted enzyme; antibody against the reaction product or thesubstance, or a labeled enzyme or substrate, or a labeled substance maybe utilized. Antibodies, enzyme, substrate, or the substance may belabeled with a detectable marker such as a radioactive label, antigensthat are recognized by a specific labeled antibody, fluorescentcompounds, enzymes, antibodies specific for a labeled antigen, andchemiluminescent compounds.

The substrate used in the method of the invention may be insolubilized.For example, it may be bound to a suitable carrier. Examples of suitablecarriers are agarose, cellulose, dextran, Sephadex, Sepharose,carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin,plastic film, plastic tube, glass beads, polyamine-methylvinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleicacid copolymer, nylon, silk, etc. The carrier may be in the shape of,for example, a tube, test plate, beads, disc, sphere etc. Theinsolubilized enzyme, substrate, or substance may be prepared byreacting the material with a suitable insoluble carrier using knownchemical or physical methods, for example, cyanogen bromide coupling.

In another aspect, the present invention includes a method for screeningfor a substance that enhances skatole metabolism by modulating thetranscription or translation of an enzyme involved in skatolemetabolism.

In one embodiment of the invention, a method is provided for screeningfor a substance that enhances skatole metabolism by enhancingtranscription and/or translation of the gene encoding aldehyde oxidasecomprising the steps of:

(a) culturing a host cell comprising a nucleic acid molecule containinga nucleic acid sequence encoding aldehyde oxidase and the necessaryelements for the transcription or translation of the nucleic acidsequence, and optionally a reporter gene, in the presence of a testsubstance; and

(b) comparing the level of expression of aldehyde oxidase, or theexpression of the protein encoded by the reporter gene with a controlcell transfected with a nucleic acid molecule in the absence of the testsubstance.

In another embodiment of the invention, a method is provided forscreening for a substance that enhances skatole metabolism by enhancingtranscription and/or translation of the gene encoding CYP2A6 comprisingthe steps of:

(a) culturing a host cell comprising a nucleic acid molecule containinga nucleic acid sequence encoding CYP2A6 and the necessary elements forthe transcription or translation of the nucleic acid sequence, andoptionally a reporter gene, in the presence of a test substance; and

(b) comparing the level of expression of CYP2A6, or the expression ofthe protein encoded by the reporter gene with a control cell transfectedwith a nucleic acid molecule in the absence of the test substance.

A host cell for use in the method of the invention may be prepared bytransfecting a suitable host with a nucleic acid molecule comprising anucleic acid sequence encoding the appropriate enzyme. Suitabletranscription and translation elements may be derived from a variety ofsources, including bacterial, fungal, viral, mammalian, or insect genes.Selection of appropriate transcription and translation elements isdependent on the host cell chosen, and may be readily accomplished byone of ordinary skill in the art. Examples of such elements include: atranscriptional promoter and enhancer or RNA polymerase bindingsequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements, such as an origin ofreplication, additional DNA restriction sites, enhancers, and sequencesconferring inducibility of transcription may be incorporated into theexpression vector. It will also be appreciated that the necessarytranscription and translation elements may be supplied by the nativegene of the enzyme and/or its flanking sequences.

Examples of reporter genes are genes encoding a protein such as greenfluorescence protein, .β-galactosidase, chloramphenicolacetyltransferase, firefly luciferase, or an immunoglobulin or portionthereof such as the Fc portion of an immunoglobulin, preferably IgG.Transcription of the reporter gene is monitored by changes in theconcentration of the reporter protein such as β-galactosidase,chloramphenicol acetyltransferase, or firefly luciferase. This makes itpossible to visualize and assay for expression of the enzyme and inparticular to determine the effect of a substance on expression ofenzyme.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,plant, or insect cells. Protocols for the transfection of host cells arewell known in the art (see, Sambrook et al. Molecular Cloning ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press,1989, which is incorporated herein by reference). Host cells which arecommercially available may also be used in the method of the invention.For example, the h2A3 and h2B6 cell lines available from GentestCorporation are suitable for the screening methods of the invention.

Substances which enhance skatole metabolism by enhancing aldehydeoxidase or CYP2A6 activity (including the substances isolated by theabove screening methods) may be used to reduce or treat boar taint or toprepare medicaments to reduce or treat boar taint.

d) Compositions

Substances which enhance skatole metabolism (including substancesidentified using the methods of the invention which selectively enhancealdehyde oxidase or CYP2A6 activity) may be incorporated intopharmaceutical compositions. Therefore, the invention provides apharmaceutical composition for use in reducing boar taint comprising aneffective amount of one or more substances which enhance skatolemetabolism and a pharmaceutically acceptable carrier, diluent, orexcipient. The term “effective amount” as used herein means an amounteffective, at dosages and for periods of time necessary to achieve thedesired result.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising an effective amount of a substance which isselected from the group consisting of

(a) a substance that increases the activity of an aldehyde oxidaseenzyme;

(b) a substance that induces or increases the expression of an aldehydeoxidase gene;

(c) a substance that increases the activity of an CYP2A6 enzyme; and

(d) a substance that induces or increases the expression of an CYP2A6gene.

The substances for the present invention can be administered for oral,topical, rectal, parenteral, local, inhalant or intracerebral use.Preferably, the active substances are administered orally (in the foodor drink) or as an injectable formulation.

In the methods of the present invention, the substances described indetail herein and identified using the method of the invention form theactive ingredient, and are typically administered in admixture withsuitable pharmaceutical diluents, excipients, or carriers suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrups and the like, consistent withconventional veterinary practices.

For example, for oral administration the active ingredients may beprepared in the form of a tablet or capsule for inclusion in the food ordrink. In such a case, the active substances can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral active substances canbe combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Suitablebinders, lubricants, disintegrating agents, and coloring agents can alsobe incorporated into the dosage form if desired or necessary. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Suitable lubricants used inthese dosage forms include sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride, and thelike. Examples of disintegrators include starch, methyl cellulose, agar,bentonite, xanthan gum, and the like.

Gelatin capsules may contain the active substance and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar carriers and diluents may be used tomake compressed tablets. Tablets and capsules can be manufactured assustained release products to provide for continuous release of activeingredients over a period of time. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration may contain coloring and flavoring agents toincrease acceptance.

Water, a suitable oil, saline, aqueous dextrose, and related sugarsolutions and glycols such as propylene glycol or polyethylene glycols,may be used as carriers for parenteral solutions. Such solutions alsopreferably contain a water soluble salt of the active ingredient,suitable stabilizing agents, and if necessary, buffer substances.Suitable stabilizing agents include antioxidizing agents such as sodiumbisulfate, sodium sulfite, or ascorbic acid, either alone or combined,citric acid and its salts and sodium EDTA. Parenteral solutions may alsocontain preservatives, such as benzalkonium chloride, methyl- orpropyl-paraben, and chlorobutanol.

The substances described in detail herein and identified using themethods of the invention can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

Substances described in detail herein and identified using the methodsof the invention may also be coupled with soluble polymers which aretargetable drug carriers. Examples of such polymers includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamidephenol,polyhydroxyethyl-aspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. The substances may also be coupledto biodegradable polymers useful in achieving controlled release of adrug. Suitable polymers include polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

Suitable pharmaceutical carriers and methods of preparing pharmaceuticaldosage forms are described in Remington's Pharmaceutical Sciences, MackPublishing Company, a standard reference text in this field.

More than one substance described in detail herein or identified usingthe methods of the invention may be used to enhance metabolism ofskatole. In such cases the substances can be administered by anyconventional means available for the use in conjunction withpharmaceuticals, either as individual separate dosage units administeredsimultaneously or concurrently, or in a physical combination of eachcomponent therapeutic agent in a single or combined dosage unit. Theactive agents can be administered alone, but are generally administeredwith a pharmaceutical carrier selected on the basis of the chosen routeof administration and standard pharmaceutical practice as describedherein.

e) Genetic Screening

The present invention further includes the identification ofpolymorphisms in genes encoding the enzymes responsible for skatolemetabolism in a pig including aldehyde oxidase and CYP2A6 as describedin detail hereinabove. The identification of genes that encode theseenzymes from pigs that are high skatole metabolizers (and hence have alow incidence of low boar taint) can be used to develop lines of pigsthat have a low incidence of boar taint. In addition, the identificationof these genes can be used as markers for identifying pigs that arepredisposed to having a low incidence of boar taint.

Accordingly, the present invention provides a method for producing pigswhich have a lower incidence of boar taint comprising selecting pigsthat express high levels of aldehyde oxidase and/or CYP2A6; and breedingthe selected pigs.

Transgenic pigs may also be prepared which produce high levels ofaldehyde oxidase and/or CYP2A6. The transgenic pigs may be preparedusing conventional techniques. For example, a recombinant molecule maybe used to introduce (a) a gene encoding aldehyde oxidase or (b) a geneencoding a CYP2A6. Such recombinant constructs may be introduced intocells such as embryonic stem cells, by a technique such as transfection,electroporation, injection, etc. Cells which show high levels ofaldehyde oxidase and/or CYP2A6 may be identified for example by SouthernBlotting, Northern Blotting, or by other methods known in the art. Suchcells may then be fused to embryonic stem cells to generate transgenicanimals. Germline transmission of the mutation may be achieved by, forexample, aggregating the embryonic stem cells with early stage embryos,such as eight cell embryos, transferring the resulting blastocysts intorecipient females in vitro, and generating germline transmission of theresulting aggregation chimeras. Such a transgenic pig may be mated withpigs having a similar phenotype i.e. producing high levels of aldehydeoxidase and/or CYP2A6 to produce animals having a low incidence of boartaint.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

Identification of Skatole Metabolites

Materials and Methods

Chemicals. 3-Methylindole (3MI), indole-3-carbinol (13C),indole-3-aldehyde, indole-3-carboxylic acid, 2-aminoacetophenone andsulfatase type H-2 from Helix pomatia were purchased from Sigma-AldrichCanada Ltd. (Oakville, ON, Canada). The oxindoles, 3-methyloxindole(3MOI) and 3-hydroxy-3-methyloxindole (HMOI) were synthesized by themethods of Kende and Hodges (1982) and Skiles et al. (1989),respectively. Authentic 5-OH-3-methylindole and 6-OH-3-methylindole (inthe form of 6-sulfatoxyskatole) were donated by Jens Hansen-Møller(Danish Meat Research Institute, Roskilde, Denmark). In order to obtain6-OH-3-methylindole from 6-sulfatoxyskatole, the compound was hydrolyzedin a total volume of 0.5 ml acetate buffer pH 5.0 containing 90 units/mlof type H-2 sulfatase. Hydrolysis was conducted for 4 hours in a shakingwater bath at 40° C. and then 0.5 ml of ice-cold acetonitrile were addedboth to stop the reaction and precipitate the protein. Aftercentrifugation at 7,500 rpm for 15 min, 50 μl of clear supernatant wereinjected into the chromatograph, using the conditions described belowunder “Analytical chromatography”.

Preparation of microsomes. Liver samples were taken from 30 intact malepigs obtained by back-crossing F3 European Wild Pig.times.SwedishYorkshire boars with Swedish Yorkshire sows (Squires and Lundström,1997). Liver samples were frozen in liquid nitrogen and stored at −80°C. For the preparation of microsomes, partially thawed liver sampleswere finely minced and homogenized with 4 volumes of 0.05 M Tris-HClbuffer pH 7.4 (containing 0.15 M KCl, 1 mM EDTA, and 0.25 M sucrose)using a Ultra-Turax homogenizer (Janke and Kunkel, GDR). The homogenatewas centrifuged at 10,000 g for 20 min and the resulting supernatant wascentrifuged again at 100,000 g for 60 min order to obtain the microsomalpellet. The pellets were suspended in a 0.05 M Tris-HCl buffer, pH 7.4,containing 20% glycerol, 1 mM EDTA, and 0.25 M sucrose to a finalconcentration of 20 mg protein/ml and stored at −80° C. before analysis.Protein concentrations were determined by the method of Smith et al.(1985) using bicinchoninic acid protein assay reagents purchased fromPierce Chemical Co. (Rockford, Ill., USA) and bovine serum albumin asstandard.

Microsomal incubations. Two mg microsomal protein was incubated with 0.4mM 3MI and 4 mM NADPH in 0.05M sodium phosphate buffer (pH 7.4)containing 5 mM MgCl₂ and 1 mM EDTA for 30 min at 37.degree. C.(production of metabolites was determined to be linear over a range of10 to 40 min). Incubation volumes were 0.5 ml. Reactions were started bythe addition of NADPH after 3-minute preincubation periods at 37° C.,and stopped with 0.5 ml of ice-cold acetonitrile. Incubations of all 30samples were run in duplicate and for control incubations NADPH wasomitted. After the addition of acetonitrile the mixture was vortexed andcentrifuged at 5000 rpm for 20 min. A 50 μl aliquot of the clearsupernatant was analyzed by high-performance liquid chromatography(HPLC).

Analytical chromatography. Analytical HPLC was done using aSpectra-Physics system (Spectra-Physics, San Jose, Calif., USA)consisting of a SP8800 gradient pump, a SP8880 autosampler with a 50 μlinjection loop, a SP Spectra 100 UV detector, and a Spectra SystemFL-2000 fluorescent detector. The HPLC method is a modification of apreviously reported binary gradient system method (Baek et al., 1995).3MI and its metabolites were separated using a reverse-phase ProdigyODS, 5 μm, 250×4.6 mm column (Phenomenex, Torrance, Calif., USA). Themobile phase consisted of two solvents, A (0.01M potassium dihydrogenphosphate buffer pH 3.9) and B (acetonitrile), with the followinggradients: 0 min—90% A, 6 min—80% A; 12 min—70% A; 18 min—30% A; 25 min10% A; 26 min 90% A; 35 min—90% A. All gradients were linear and theflow rate was set at 1.2 ml/min. Absorbance was monitored at 250 nm;fluorescence was monitored at excitation and emission wavelengths of 286and 350 nm, respectively. HPLC analysis for 3MI metabolites wasconducted immediately after the incubations. Metabolites were identifiedby comparison of retention times, and co-injection of standards (spikingthe metabolite mixture with authentic standards).

Isolation and purification of metabolites by preparative HPLC. In orderto obtain a sufficient amount of metabolites to conduct UV spectralanalysis, a large scale incubation (final volume of 4 ml) was performed,using the same concentrations of reactants as described above.Preparative HPLC was done using a Spectra-Physics SP8800 gradient pump(Spectra-Physics, San Jose, Calif., USA), a manual Rheodyne 7125injector fitted with a 500 μl injection loop (Rheodyne, Cotati, Calif.,USA), and a SP Spectra 100 UV detector. The 3MI metabolites wereseparated using a reverse-phase Waters preparative HPLC C18, 10 μm,300.times.7.6 mm column (Waters Associates, Division of Millipore Corp.,Milford, Mass., USA). The mobile phase was the same as above except thatthe flow rate was set at 3.0 ml/min. The peaks corresponding to themetabolites identified on the basis of their retention times as HMOI,I3C, 3MOI and 2-aminoacetophenone were collected in enough amounts todetermine their UV spectra. Purity of the collected fractions wasverified by HPLC using the procedure described before under “Analyticalchromatography”. One of the metabolites produced by pig liver microsomescould not be identified on the basis of comparison of retention times;this metabolite was named UV-1 due to its absorption in the far UVspectrum and the fact that it was the first metabolite that eluted fromthe column (Babol et al., 1998a). The peak corresponding to thismetabolite, which eluted between 9.1 and 10.1 min, was collected afterseveral 500 μl injections and subjected to HPLC-MS, sup.1H-NMR and UVspectra analysis.

Ultraviolet Spectroscopy. UV spectra (200-300 nm) were recorded for theHPLC metabolites UV-1, HMOI, I3C, 3MOI and 2-aminoacetophenone. UVspectra of available authentic standards were also recorded and comparedwith those of the isolated metabolites. Spectra were recorded on a model4054 LKB Biochrom UV-Visible spectrophotometer (Pharmacia LKB BiochromLtd. Cambridge, UK). Due to their low levels of production, it was notpossible to isolate the hydroxyskatoles in enough quantities todetermine their UV spectra.

LC/MS of metabolite UV-1. Metabolite UV-1 was analyzed by LC-MS usingthe following conditions: the HPLC was performed using a Prodigy 5ODS-2, 5 μm, 150.times.3.2 mm column (Phenomenex, Torrance, Calif., USA)and water:acetonitrile (50:50) as mobile phase. The mobile phase wasdelivered by binary LC pumps (Hewlett Packard 1090 Series II/L, PaloAlto, Calif., USA). The eluent passed through a sample injection valveRheodyne 7010 (Rheodyne, Cotati, Calif, USA), to an atmospheric pressurechemical ionization (APCI) source configured with a corona dischargepin, at a flow rate of 0.7 ml/min. A sample volume of 20 μl was injectedby an autosampler (Hewlett Packard 1090 Series II/L, Palo Alto, Calif.,USA). Mass spectrometry (MS) detection was achieved using a VG QuattroII triple quadrupole mass spectrometer (Fisons UK Ltd., Altrincham, UK).Instrument control, data acquisition and data processing were carriedout using the MassLynx software package. Liquid nitrogen was used as adrying and sheath gas, at flow rates of 200 and 50 liter/hr,respectively. The instrument was operated in the positive ion mode withan ion source temperature of 150° C., a corona discharge pin potentialof +3.75 kV, and a cone voltage of 15V. The total ion chromatogram ofLC/MS was obtained by scanning the first quadrupole from m/z 125-700 ata rate of 400 amu/sec in full scan mode with inter-scan delay of 0.10sec. Data was acquired in continuum mode. The production scan wasperformed by tandem mass spectrometry (MS/MS) by transmitting theprotonated molecular ion ([M+H].sup.+) through the first quadrupole intothe second quadrupole containing ultrapure argon. The productionchromatogram was recorded by scanning the third quadrupole from m/z 50to 450 in 1.0 sec. The collision energy was varied between −20 to −50 eVto optimize fragmentation of the selected protonated molecular ion.

NMR spectroscopy of metabolite UV-1. UV-1 metabolite was isolated forNMR analysis using incubation conditions essentially as described above.However, these incubations contained 1 nmol cytochrome P450 contentrather than 2 mg of total protein. UV-1 was separated from othermicrosomal 3MI metabolites by the HPLC conditions described above usinga system consisting of an LDC Analytical Constametric 4100 solventdelivery module (ThermoQuest, Riviera Beach, Fla., USA), a HewlettPackard 1040A diode array detector and a Hewlett Packard 9000 seriesHPLC workstation (Hewlett Packard Company, Willington, Del., USA). WV-1was purified by HPLC and pooled from two identical incubations followedby concentration in a Savant Speed-Vac (Savant Instruments, Farmingdale,N.Y., USA). Concentration to dryness was not possible, due topolymerization and degradation of unstable UV-1. Therefore, the samplewas evaporated to a volume of 200 L and re-injected on the HPLC foradditional purification. In this case however, the aqueous mobile phaseconsisted of 0.01 M dibasic potassium phosphate buffer, pH 9.0, in 99.9atom % deuterium oxide. Due to the instability of UV-1 when it wasevaporated to dryness, it was necessary to perform the finalpurification step in the NMR solvent, deuterium oxide. UV-1 was againcollected and evaporated to a final volume of 250 L and directly addedto the Shigemi NMR tube. The .sup.1 H-NMR spectrum was obtained indeuterium oxide using a Varian Unity Inova 600 MHz NMR (VarianAssociates Inc., Palo Alto, Calif, USA).

Results

HPLC. None of the metabolites produced by pig liver microsomes co-elutedwith indole-3-carboxaldehyde or indole-3-carboxylic acid. However,metabolites that coeluted with HMOI, 3MOI, I3C, 2-aminoacetophenone, andthe two hydroxyskatoles (5- and 6-OH-3-methylindole) were measured by UVand/or fluorescence detection. The oxindole metabolites (HMOI and 3MOI)and the pyrrole ring opened metabolite (2-aminoacetophenone) weredetected and quantitated by UV absorption because they do not fluoresce;I3C and the hydroxyskatoles were detected and quantitated byfluorescence detection. When microsomal incubations were spiked, allmetabolites identified on the basis of their retention times,co-chromatographed with their corresponding authentic standards. Thechromatographic profile of a microsomal incubation and a standardmixture monitored by UV absorption at 250 nm is shown in FIG. 1.

UV Spectroscopy. The UV spectrum of the metabolites identified on thebasis of their retention times on HPLC (HMOI, 3MOI, 13C, and2-aminoacetophenone) were identical to those of authentic standards.Spectra of metabolites were recorded using water as solvent, and thewavelengths of maximal absorption were as follows: HMOI: λ_(max) (nm):208, 253; 3MOI: λ_(max) (nm): 205, 252; 13C: λ_(max) (nm): 221, 278;2-aminoacetophenone: λ_(max) (nm): 228, 257. The UV spectrum of3-methylindole was: λ_(max) (nm): 224, 281. The UV spectrum of UV-1metabolite was: λ_(max) (nm): 204, 238. The UV spectra of UV-1 wassimilar to the spectra of the oxindole metabolites 3MOI and HMOI asshown in FIG. 2. Changing the pH from 3 to 11 did not change thespectrum of UV-1; this lack of a bathochromic shift indicated that theunknown metabolite had no free phenolic group. Isolated UV-1 was kept inacetonitrile:water solution at room temperature and the solution wasanalyzed by HPLC at 7-day intervals for 6 weeks. After 6 weeks onlyabout 25% of the original compound remained and it was observed thatUV-1 was converted into 3MOI. The slopes of the linear regressions of3MOI and UV-1 over time indicated that the molar response factor forUV-1 on HPLC-UV analysis was 2.95 times that of 3MOI.

Metabolite UV-1 structural data. The mass spectrometry of isolated UV-1produced a molecular ion at m/z 148 [M+H].sup.+ with major fragments atm/z 133 [M-CH₃]⁺, 104 [M-H₃ C—C—OH]⁺, and 77 protonated phenyl ring)(FIG. 3). The ¹H-NMR spectrum of metabolite UV-1 is shown in FIG. 4.Assignments of the proton signals are provided, listed as chemical shift(multiplicity, integration and assignment): 1.4 (s, 3H, —CH₃); 6.8 (d,2H, H-5 and H-6); 7.2 (d, 2H, H-4 and H-7); 8.4 (s, 1H, H-2). Thesinglet at 8.4 has been assigned to the proton at C-2 of3-hydroxy-3-methylindolenine. This proton is attached to the sp.sup.2hybridized C-2 which is also a deshielded by the adjacent nitrogen.Therefore, this proton is highly deshielded and appears downfield fromall other protons in the proposed structure. At 2.0 is a singletcorresponding to the methyl protons of contaminating acetonitrile. Dueto the way in which the sample was purified, it was extremely difficultto remove all of the acetonitrile present in the HPLC organic phase.

In summary, seven metabolites of 3MI were found to be produced by pigliver microsomes: 3MOI, HMOI, 6-OH-3-methylindole (6-OH-3MI), I3C,2-aminoacetophenone, 5-OH-3-methylindole (5-OH-3M]), and the metabolitethat was named UV-1. When UV-1 was quantitated assuming a molarabsorptivity 2.95 times greater than that of 3MOI, the total amount ofnanomoles produced accounted for an average of 96.0% (range of86.5-105.0%) of the 3MI molecules metabolized during the microsomalincubations. The rates of production of the seven metabolites identifiedin pig liver microsomal incubations are shown in Table 1. UV-1metabolite was produced at the highest rate (750.7 pmol/mg protein/min),while 5-OH-3MI was produced at the lowest rate (5.1 pmol/mgprotein/min). Large inter-individual differences were noted for theproduction rates of the same metabolite. For instance, UV-1 metabolitewas produced at a rate of 1556.3 pmol/mg protein/min by the microsomesof one pig, while other microsomes produced this compound at a rate of180.5 pmol/mg/protein/min (Table 1). The metabolite that was produced inlarger amounts was UV-1 which, on average, accounted for 45.1% of allmetabolites produced. The combined oxindoles accounted for 46.4% of thetotal metabolites: an average of 27.9% of the metabolites producedcorresponded to 3MOI whereas 18.5% corresponded to HMOI. The othermetabolites were produced in much lesser amounts. 6-OH-3MI accounted for4.9% of the metabolites, I3C accounted for 2.7% and 2-aminoacetophenoneand 5-OH-3MI accounted for only 0.5% and 0.3% of the metabolites,respectively. The chemical structures and percentages of production ofthese metabolites are shown in FIG. 5.

Discussion

Only three Phase I metabolites of 3MI had been identified previously inpigs: HMOI, and the hydroxyskatoles, 5-OH-3MI and 6-OH-3MI. HMOI hadbeen found in pig plasma and urine (Baek et al., 1997), and pig livermicrosomal incubations (Babol et al., 1998a); 6-OH-3MI had been detectedboth in pig serum (Baek et al., 1997) and pig liver microsomalincubations (Babol et al., 1998a), while 5-OH-3MI had only been reportedto be present in pig serum (Baek et al., 1997). In the present study,all three metabolites were detected in the microsomal incubations andthe production of four new metabolites is reported.

One of the pathways of 3MI biotransformation identified in species suchas goats, mice and rats is the formation of oxindole derivatives: 3MOIand HMOI (Frydman et al., 1972; Smith et al., 1993). On average, 46.4%of the metabolites produced by pig liver microsomes in the present studycorresponded to these two oxindole derivatives; this finding indicatesthat the oxidole pathway is quantitatively very important in the pig.3MOI had been identified in rat liver microsomal incubations (Frydman etal., 1972), goat lung and liver microsomal incubations (Huijzer et al.,1987), and in the urine of goats (Hammond et al., 1979). One of themetabolites observed in pig microsomal incubations by Babol et al.(1998a) was named “UV-3” and the results of the present study indicatethis metabolite corresponds to 3MOI. The other oxindole derivative of3MI, HMOI, had already been isolated from the urine of pigs dosed with3MI (Baek et al., 1997) and was also reported to be produced by pigliver microsomes (Babol et al., 1998a); HMOI is also a major urinarymetabolite produced by mice dosed with radiolabeled 3MI (Skiles et al.,1989), additionally it has been found in the urine of humans (Albrechtet al., 1989), and goats (Smith et al., 1993). Interestingly, in thepresent study, pig liver microsomes produced large amounts of bothoxidole derivatives 3MOI and HMOI. In other species studied, one ofthese metabolites predominates. In goats, production of 3MOIpredominates (Hammond et al., 1979), whereas in mice it is H4MOI thatpredominates (Smith et al., 1993).

The 3 methyl group of 3MI may be oxidized to the alcohol, aldehyde andcarboxylic acid functions (Hammond et al., 1979). In the present study,only the alcohol function of the 3 methyl group (indole-3-carbinol) wasfound to be produced by pig liver microsomes. This metabolite exhibitsstrong fluorescence and also absorbs in the UV and even though it hadbeen previously reported to be produced by pig microsomes (named F-1 byBabol et al., 1998a), its structure was unknown. It is important to notethat further metabolism of the alcohol function of indole-3-carbinolcould possibly be catalyzed by alcohol dehydrogenase; if this is true,then the product of this reaction, indole-3-carboxaldehyde, would not beproduced in microsomal incubations.

Hydroxylation of the aromatic ring of 3MI can occur at any of thecarbons 4, 5, 6 or 7; however, the experimental evidence indicates thathydroxylation at positions 5 and 6 predominate. In 1962, Jepson andco-workers showed that rabbit liver microsomes hydroxylate tryptamine,indole acetic acid and related indoles to their corresponding 6-hydroxyderivatives. The microsomal system required NADPH and oxygen and did notform 5- or 7-hydroxyindoles (Jepson et al., 1962). Mahon and Mattok(1967) analyzed the urine of ten normal human subjects and found thatall samples contained 6-hydroxyskatole and nine had the 5-isomer,although its excretion rate was approximately 50% of the 6-isomer;7-hydroxyskatole was detected in three of the samples but its excretionrate was only 5% of the 6-isomer. None of the subjects excreted4-hydroxyskatole (Mahon and Mattok, 1967). Baek et al. (1995) foundconjugates of both 5-OH-3MI and 6-OH-3MI in pig serum. In the presentstudy, the average rate of production of 6-OH-3MI was approximatelyeleven times greater than the production of the 5 isomer, indicatingthat hydroxylation at position C6 predominates.

Frydman et al. (1972) found two pyrrole ring opened metabolites producedafter incubation of 3-MI with rat liver microsomes. The two compoundswere identified as 2-formamidoacetophenone and 2-aminoacetophenone; atotal of 33% of the metabolites formed corresponded to2-formamidoacetophenone, 12% to 2-aminoacetophenone, and 5% to 3-MOI. Inthe present study, 2-aminoacetophenone was found to be produced by allliver samples analyzed at an average percentage of 0.5%, which is muchlower than the percentage reported for rats by Frydman et al. (1972). Noprevious reports of 2-aminoacetophenone production from 3MI metabolismby pigs were found in the literature.

The ¹H-NMR, LC-MS and UV-spectral characteristics of metabolite UV-1indicate that this compound corresponds to 3-hydroxy-3-methylindolenine.UV-1 was found to be an unstable compound, intermediate between 3M1 and3MOI. The fact that UV-1 was converted into 3MOI suggested that thiscompound could be a precursor of 3MOI, possibly2,3-epoxy-3-methylindolenine, the structure of which was postulated bySmith et al. (1993) or, most likely, its ring-opened product,3-hydroxy-3-methylindolenine (Skordos et al., 1998a, 1998b). Themolecular weight of the compound (147) and its fragmentation patternwere compatible with the epoxyde or the imine (FIG. 3), but the UVspectrum, with a λ_(max) at 238 nm (FIG. 2) was more consistent with theimine structure. The molecular weight of 147 could also correspond to anaromatic phenolic metabolite of 3MI; however, when the UV spectrum ofisolated UV-1 was taken under different pHs, it did not show the typicalbathochromic shift observed in phenolic indoles. Furthermore, the factthat the UV spectrum of metabolite UV-1 was very similar to that of 3MOIand HMOI (FIG. 2) indicated that metabolite UV-I could be structurallyrelated to any of the two oxindoles; these metabolites, in which thepyrrol ring is oxidized at the 2-carbon position, show very differentspectra than 3MI, or other metabolites such as I3C, 2-aminoacetophenoneor the hydroxyskatoles. Finally, the ¹H-NMR spectrum of UV-1 (FIG. 4)was consistent with the assignment of this metabolite to3-hydroxy-3-methylindolenine.

The results of the present study indicate that seven major metabolitesof 3MI are produced by pig liver microsomes in vitro. In quantitativeterms, the main pathway of Phase I biotransformation of 3MI by pig livermicrosomes appears to be the formation of oxindole derivatives and theformation of 3-hydroxy-3-methylindolenine. Differences in the metabolicfate of 3MI among species could explain the difference in speciessusceptibility to 3Ml-induced lung toxicity. The extensive metabolism of3MI to oxindole derivatives may explain the lack of pneumotoxicityshowed by pigs and reported by Carlson and Yost (1989). Theelectrophilic metabolite 3-methylene-indolenine, which is the putativereactive metabolite of 3MI produced by cytochrome P450 enzymes, is aprecursor of I3C in lung microsomal incubations and susceptible speciesform I3C in appreciable amounts (Skiles and Yost, 1996). In the presentin vitro study, less than 3% of the metabolites produced by pig livermicrosomes corresponded to I3C, which may also explain the lack ofsusceptibility of pigs to suffer from 3MI-induced lung lesions. Largeinter-individual differences in the rate of production of metaboliteswere observed. These differences in Phase I metabolism could be due toindividual differences in cytochrome P450 enzymes and this issue shouldbe further investigated. It was previously reported that CYP2E1 plays arole in the metabolism of 3M1 in the pig (Squires and Lundström, 1997;Babol et al., 1998a), but the role of other isoenzymes remains to bedetermined. Babol et al. (1998b) reported sulfation and glucuronidationof some 3MI metabolites produced by pig liver microsomes. However, morestudies are needed in order to determine the complete Phase IImetabolism of the different metabolites of 3MI identified in the presentstudy.

Example 2

Aldehyde Oxidase

Materials And Methods

Chemicals. Menadione, quinacrine and allopurinol were purchased fromSigma-Aldrich Canada (Oakville, ON, Canada). Authentic HMOI wasgraciously provided by Dr. G. S. Yost, Department of Pharmacology andToxicology, University of Utah. HMI was produced using porcine livermicrosomes and it was isolated and purified using preparative HPLC asdescribed before (Diaz et al., 1999). Isolated HMI was freeze-dried andkept in a dessicator at −20° C. until used.

Preparation of porcine liver cytosol. Liver samples were taken from 30intact male pigs obtained by back-crossing F3 European Wild Pig×SwedishYorkshire boars with Swedish Yorkshire sows (Squires and Lundström,1997). Liver samples were frozen in liquid nitrogen and stored at −80°C. For the preparation of the cytosolic fraction, partially thawed liversamples were finely minced and homogenized with 4 volumes of 0.05 MTris-HCl buffer pH 7.4 (containing 0.15 M KCl, 1 mM EDTA, and 0.25 Msucrose) using a Ultra-Turax homogenizer (Janke and Kunkel, GDR). Thehomogenate was centrifuged at 10,000×g for 20 minutes and the resultingsupernatant was centrifuged again at 100,000×g for 60 minutes in orderto obtain the cytosolic fraction and the microsomal pellet. Cytosolicfractions were stored at −80° C. before analysis. Protein concentrationswere determined by the method of Smith et al. (1985) using bicinchoninicacid protein assay reagents purchased from Pierce Chemical Co.(Rockford, Ill., USA) and bovine serum albumin as standard.

Enzyme assays. In order to investigate the role of AO in the conversionof HMI to HMOI, incubations containing HMI, porcine liver cytosol anddifferent concentrations of the selected AO inhibitors menadione andquinacrine were conducted. Each incubation was run in duplicate, andwere performed for three randomly selected cytosol porcine samples.HIMOI formation was detected and quantitated by HPLC as described under“Chromatographic analysis”. AO activity was measured as the formation ofHMOI per minute per mg of cytosolic protein. Assay mixtures contained0.05M sodium phosphate buffer (pH 7.4) with 5 mM MgCl₂ and 1 mM EDTA, 1mg cytosolic protein and 1 μg HMI in a final assay volume of 250 μl. Forthe inhibition experiments, different final concentrations of menadione(0, 2, 5, 10, 25, 50 and 100 μM) or quinacrine (0, 0.05, 0.1, 0.25, 0.5and 1.0 mM) were tested in the assay mixture. Menadione was dissolved inethanol (final assay concentration 4%, v/v), which had no effect onactivity in controls without inhibitor; quinacrine was dissolved inbuffer. Incubations were carried out for 10 min at 37° C. in a shakingwater bath; the reaction was stopped with 250 μl ice-cold acetonitrile.After the addition of acetonitrile, the mixture was vortexed andcentrifuged at 7,500 rpm for 15 min. A 400 μl aliquot of the clearsupernatant was diluted with 400 μl water and 100 μl of the mixture wereanalyzed immediately by high-performance liquid chromatography (HPLC).Dilution with water was necessary in order to avoid leading of thechromatographic peaks. HMOI production was quantitated by using anexternal standard. Controls included incubations using boiled cytosoland incubations carried out without the addition of cytosol. Incubationsrun under the same conditions described above were conducted using 0.1,0.5 and 1.0 mM allopurinol in order to investigate the role of XO on theenzymatic conversion of HMI into HMOI.

Chromatographic analysis. HPLC was conducted using a Spectra-Physicssystem (Spectra-Physics, San Jose, Calif, USA) consisting of a SP8800gradient pump, a SP8880 autosampler with a 100 μl injection loop, and aSP Spectra 100 UV detector. The HPLC method is a modification of apreviously reported binary gradient system method (Baek et al., 1997).HMOI and HMI were separated using a reverse-phase Prodigy ODS, 5 μM,250×4.6 mm column (Phenomenex, Torrance, Calif., USA). The mobile phaseconsisted of two solvents, A (0.01M potassium dihydrogen phosphatebuffer pH 3.9) and B (acetonitrile), with the following gradients: 0min—90% A, 6 min—80% A; 12 min—70% A; 18 min—30% A; 25 min 10% A; 26 min90% A; 35 min—90% A. All gradients were linear and the flow rate was setat 1.2 ml/min. Absorbance was monitored at 250 um. HPLC analysis wasconducted immediately after the incubations.

Measurement of 3MI fat content. Por the quantitation of the 3MI fatcontent, a sample of backfat was taken from each pig and its 3MI contentmeasured with a colorimetric assay (Mortensen and Sorensen, 1984). Allanalysis were done in duplicate.

Statistical analysis. Pearson correlation coefficients, linearregression analysis and one-way ANOVA were computed using theStatistical Analysis System (SAS, 1995).

Results

Porcine cytosol catalyzed the conversion of HMI to HMOI (FIG. 6) in atime-dependent manner (FIG. 7). Under these assay conditions, theformation of HMOI was found to be linear (r²=0.995) up to 10 min (FIG.7). No HMOI was formed when cytosol was boiled before the incubation orwhen no cytosol was added to the assay mixture. The addition of thealdehyde-oxidase inhibitors menadione or quinacrine to the incubationmixtures containing HMI and cytosolic protein decreased the formation ofHMOI in a dose-dependent manner. When no inhibitor was added, the totalamount of HMOI produced was considered as 100%. At a concentration of 10μM menadione, only 33.3% of the HMOI formed in the absence of menadionewas detected whereas at a concentration of 100 μM menadione, no HMOI wasproduced (FIG. 8). At a concentration of 50 A quinacrine, 75.5% of thecontrol HMOI production was observed and at 1 mM 43.4% of the controlHMOI was found (FIG. 9). Menadione was a more potent inhibitor of thereaction since even a concentration of quinacrine 10 times higher thanthat of menadione (1 mM vs 100 μM) was not enough to completely abolishthe conversion of HMI to HMOI. The addition of up to 1.0 mM allopurinolto the assay mixture did not affect the conversion of HMI to HMOI (datanot shown).

The AO activity, estimated as nmol of HMOI produced per minute per mgcytosolic protein, versus the 3MI fat content of the 30 pigs used inthis study are shown in FIG. 10. The Pearson correlation coefficientbetween these two variables was found to be −0.70 (P<0.001), whereas thedetermination coefficient was r²=0.49. The linear regression model toexplain the 3MI fat content as a function of AO activity was found tobe: 3MI in fat=0.22−AO activity 0.042763. Tis model was found to behighly significant (P<0.001).

The 3MI fat content in all samples ranged from 0.07 to 0.3 mg/kg and hadmean value of 0.15 mg/kg, whereas the AO activity ranged from 0.25 to3.53 nmol HMOI/mg protein/min and had a mean value of 1.27 nmol HMOI/mgprotein/min. The results were grouped in three categories according tothe 3MI fat content of each pig as follows: large 3MI accumulators (0.2mg/kg 3MI or more), moderate 3M1 accumulators (0.1 1 to 0.19 mg/kg 3MI)and low accumulators (0.1 mg/kg 3MI or less). Lundström and Bonneau(1996) have suggested that levels of 3M1 of 0.2-0.25 mg/kg or greatercause unacceptable taint by sensory analysis. The mean values for 3MIfat content and AO activity for these three categories of pigs are shownin Table 2.

Discussion

Menadione is a well documented inhibitor of AO (Johns, 1967; Krenitzy etal., 1974; Rodrigues, 1994) and biochemical reactions sensitive toinhibition by menadione are attributed to AO (Beedham et al., 1995;Rashidi et al., 1997). Rodrigues (1994) found that at a concentration of10 μM, menadione completely abolished the oxidation ofN¹-methylnicotinamide, the model substrate for AO. In the presentexperiment, a concentration of 10 μM menadione decreased the formationof HMOI by 56.7%, and at 100 μM menadione, no HMOI was formed,indicating a complete inhibition of the enzymatic activity. The inversedose-response relationship observed between HMOI production andmenadione concentration strongly suggests that AO is the enzymeresponsible for the biotransformation of HMI into HMOI in porcinecytosol. Quinacrine has been reported as being a competitive inhibitor(K_(i)=1.5.times.10⁻⁶ M) of aldehyde oxidase against all substrates(Rajagopalan and Handler, 1964). In the present trial, quinacrine wasless potent than menadione in inhibiting the conversion of HMI into HMOIbut it also inhibited the reaction to a large extent. The inhibition ofHMOI formation caused by quinacrine also suggests that the production ofHMOI from HMI is catalyzed by AO. On the other hand, the lack ofinhibition observed when allopurinol was added to the reaction mixtureindicates that XO is not involved in the oxidative metabolism of HMIinto HMOI.

N-heterocyclic cations constitute a major group of substrates for AO(Beedham, 1985). Quaternization of a ring nitrogen atom activates theheterocycle to nucleophilic substitution and enhances the reactivity ofthe compound toward enzyme-catalyzed attack (Beedham, 1985). HMI is arecently identified N-heterocyclic quaternized metabolite produced byporcine microsomal enzymes (Diaz et al., 1999) and therefore itconstitutes a suitable substrate for AO-catalyzed oxidation. The resultsof the present study strongly suggest that AO activity present in thecytosol of pigs is responsible for the oxidation of HMI to form a morepolar and stable metabolite, HMOI.

When hepatic AO activity (measured as the formation of HMOI) was plottedagainst the 3MI fat content, a clear inverse relationship was observed(FIG. 9). This finding suggests that hepatic AO activity is related to3MI clearance. The relatively high determination coefficient (r²=0.49)indicates that almost 50% of the variation in 3MI fat content isexplained by the hepatic enzymatic activity of AO. The results shown onTable 2 also indicate that AO activity may be very significant in theadequate clearance of 3MI in the pig. High 3MI fat levels wereassociated with low enzymatic activity (mean values of 0.24 mg/kg 3MIand 0.80 nmol HMOI/mg protein/min, respectively), whereas low 3MI levelswere associated with high enzymatic activity (mean values of 0.09 mg/kg3MI and 2.73 nmol HMOI/mg protein/min, respectively). Pigs classified ashigh 3MI accumulators had a hepatic mean-AO activity 3.4 times lowerthan those pigs classified as low accumulators; this difference wasfound to be significant (P<0.05).

The results of the present study suggest that AO plays an important rolein the metabolism of 3MI in the pig and that its catalytic activity isrelated to an adequate 3MI clearance. The enzymatic activity of AO inthe pig might be used as a potential marker in order to identify pigscontaining low levels of 3MI in the fat, which will eventually help tocontrol “boar taint”.

Menadione is customarily used as a source of vitamin K in swine diets(National Research Council, 1987). ecommended levels of inclusion are2.5 mg/kg for grower diets and 2.0 mg/kg for finisher diets (Patience etal., 1995). Since menadione is a potent inhibitor of AO and the enzymeappears to be important in the metabolism of 3MI, care should beexercised so that excessive levels of menadione are not present in swinediets. It is possible that some of the sporadic episodes of “boar taint”could had been caused by high levels of menadione in the diet resultingin high levels of 3MI in the fat of pigs. Studies are needed in order todetermine whether the levels of menadione commonly used in practical pigdiets are capable of inhibiting AO activity. Additionally, it has beenobserved that high levels of dietary copper lead to molybdenumdeficiency and thus to low AO activity because molybdenum is a cofactorfor this enzyme (Beedham, 1985). It is important to avoid excess copperlevels in pig diets in order to avoid a decrease in the activity of AOand the potential occurrence of “boar taint” episodes.

Example 3

The Role of CYP2A6 IN 3-Methylindole Metabolism by Porcine LiverMicrosomes

The role of different cytochrome P450 enzymes on the metabolism of3-methylindole (3MI) was investigated using selective chemicalinhibitors. Eight chemical inhibitors of P450 enzymes were screened fortheir inhibitory specificity towards 3MI metabolism in porcinemicrosomes: alpha-naphthoflavone (CYP1A2), 8-methoxypsoralen (CYP2A6),menthofuran (CYP2A6), sulphaphenazole (CYP2C9), quinidine (CYP2D6),4-methylpyrazole (CYP2E1), diethyldithiocarbamate (CYP2E1, CYP2A6), andtroleandomycin (CYP3A4). The production of the different 3MI metaboliteswas only affected by the presence of inhibitors of CYP2E1 and CYP2A6 inthe microsomal incubations. In a second experiment, a set of porcinemicrosomes (n=30) was screened for CYP2A6 content by Western blotanalysis and also for their 7-hydroxylation activity (CYP2A6 activity).Protein content and enzymatic activity were found to be correlated with3MI fat content. The results of the present study indicate thatmeasurement of CYP2A6 levels and/or activity is a useful marker for3MI-induced boar taint. TABLE 1 Rate of production of 3MI metabolites bypig liver microsomes (pmol/mg microsomal protein/min) (n = 30) Rate ofProduction Minimum Maximum (pmol/mg prot./ (pmol/mg (pmol/mg Metabolitemin) ± SD prot./min) prot./min) UV-1 750.7 ± 414.5 180.5 1556.33-methyloxindole 420.9 ± 118.1 234.4 700.8 3-hydroxy-3- 272.4 ± 91.6 118.9 516.5 methyloxindole 6-OH-3-methylindole 58.4 ± 47.2 n.d.* 213.7Indole-3-carbinol 37.1 ± 15.8 12.1 85.7 2-aminoacetophenone 7.8 ± 2.43.4 12.7 5-OH-3-methylindole 5.1 ± 5.8 0.7 27.3*n.d. = not detected

TABLE 2 Mean (SD) Mean (SD aldehyde 3-methylindole oxidas activity3-Methylindole content (nmol HMOI/ Category fat content n (mg/kg) mgprot./min High 0.2 mg/kg or 7 0.24 0.4^(a) 0.80 0.61^(b) accumulatormore Moderate 0.11-0.19 15 0.15 0.03^(b) 1.40 0.90^(b) accumulator mg/kgLow 0.1 mg/kg or 8 0.09 0.01^(c) 2.73 0.45^(a) accumulator less^(a-c)Within a column, means lacking a common superscript differsignificantly (P < 0.05).

Example 4

According to the invention, the association of alternate forms ofcytochrome P450 enzymes such as the CYP2A6 may be used to identify andselect pigs with differences in boar taint. For example, according tothe invention, a deletion mutant of the CYP2A6 gene has been identifiedthat results in a frame shift and loss of function mutation, whichresulted in higher skatole levels in the pig.

We have cloned the pig isoforms of CYP2A6. We found a deletion mutationthat results in a frame shift and premature stop. This animal has zeroenzyme activity for CYP2A6 (coumarin 7-hydroxylase) in the liver andhigh skatole levels in fat. Another polymorphism was identified whichresulted in a t to c transition at nt number 124 and a change from Pheto Leu at amino acid number 42 of SEQ ID NO:3 (wild type).

Further according to the invention, other polymorphisms in genes relatedto skatole metabolism (other cytochrome P450 related genes) in the pigmay be identified to genetically identify and select pigs based upontheir proclivity to boar taint. Once an association between a gene orgene product and a particular trait is made, genes encoding theseproteins may be screened for polymorphism or markers which may be usedto indicate differences in these animals with respect to the correlatedtrait. These polymorphisms with these genes enables genetic markers tobe identified for specific breeds or genetic lines or animals, boartaint potential early in the animal's life.

An alternate form of CYP2A6 has been identified according to theinvention which results in a frameshift causing a premature stop codonand loss of function resulting in higher skatole levels in the pig.Tests for the presence of this alternate form may be developed using thenovel sequence for CYP2A6 as disclosed herein, SEQ ID NO: 18 or 3 supraand the mutations disclosed herein in SEQ ID NO:l (both 124 nt and 422deletion), SEQ ID NO:5 (124nt only) and SEQ ID NO:7 (422 deletion only).These tests include but are not limited to PCR, SSCP, and the like.

The invention thus relates to genetic markers for economically valuabletraits in animals. The markers represent alleles or alternate gene formsthat are associated boar taint, based upon the findings that thealdehyde oxidase pathway and CYP2A6 are associated with skatoleproduction.

Thus, the invention relates to genetic markers and methods ofidentifying those markers in an animal of a particular animal, breed,strain, population, or group, whereby the animal is has increased,decreased or otherwise altered skatole metabolism, and thus boar taint.

Any method of identifying the presence or absence of these markers maybe used, including, for example, single-strand conformation polymorphism(SSCP) analysis, base excision sequence scanning (BESS), RFLP analysis,heteroduplex analysis, denaturing gradient gel electrophoresis, andtemperature gradient electrophoresis, allelic PCR, ligase chain reactiondirect sequencing, mini sequencing, nucleic acid hybridization,micro-array-type detection of genes encoding enzymes involved in skatolemetabolism. Also within the scope of the invention includes assaying forprotein conformational or sequences changes which occur in the presenceof this polymorphism. The polymorphism may or may not be the causativemutation but will be indicative of the presence of this change and onemay assay for the genetic or protein bases for the phenotypicdifference.

The following is a general overview of techniques which can be used toassay for the genetic marker of the invention.

In the present invention, a sample of genetic material is obtained froman animal. Samples can be obtained from blood, tissue, semen, etc.Generally, peripheral blood cells are used as the source, and thegenetic material is DNA. A sufficient amount of cells are obtained toprovide a sufficient amount of DNA for analysis. This amount will beknown or readily determinable by those skilled in the art. The DNA isisolated from the blood cells by techniques known to those skilled inthe art.

Isolation and Amplification of Nucleic Acid

Samples of genomic DNA are isolated from any convenient source includingsaliva, buccal cells, hair roots, blood, cord blood, amniotic fluid,interstitial fluid, peritoneal fluid, chorionic villus, and any othersuitable cell or tissue sample with intact interphase nuclei ormetaphase cells. The cells can be obtained from solid tissue as from afresh or preserved organ or from a tissue sample or biopsy. The samplecan contain compounds which are not naturally intermixed with thebiological material such as preservatives, anticoagulants, buffers,fixatives, nutrients, antibiotics, or the like.

Methods for isolation of genomic DNA from these various sources aredescribed in, for example, Kirby, DNA Fingerprinting, In Introduction,W.H. Freeman & Co. New York (1992). Genomic DNA can also be isolatedfrom cultured primary or secondary cell cultures or from transformedcell lines derived from any of the aforementioned tissue samples.

Samples of animal RNA can also be used. RNA can be isolated from tissuesexpressing the gene as described in Sambrook et al., supra. RNA can betotal cellular RNA, mRNA, poly A+ RNA, or any combination thereof. Forbest results, the RNA is purified, but can also be unpurifiedcytoplasmic RNA. RNA can be reverse transcribed to form DNA which isthen used as the amplification template, such that the PCR indirectlyamplifies a specific population of RNA transcripts. See, e.g., Sambrook,supra, Kawasaki et al., Chapter 8 in PCR Technology, (1992) supra, andBerg et al., Hum. Genet. 85:655-658 (1990).

PCR Amplification

The most common means for amplification is polymerase chain reaction(PCR), as described in U.S. Pat. Nos. 4,683,195; 4,683,202; and4,965,188 each of which is hereby incorporated by reference. If PCR isused to amplify the target regions in blood cells, heparinized wholeblood should be drawn in a sealed vacuum tube kept separated from othersamples and handled with clean gloves. For best results, blood should beprocessed immediately after collection; if this is impossible, it shouldbe kept in a sealed container at 4° C. until use. Cells in otherphysiological fluids may also be assayed. When using any of thesefluids, the cells in the fluid should be separated from the fluidcomponent by centrifugation.

Tissues should be roughly minced using a sterile, disposable scalpel anda sterile needle (or two scalpels) in a 5 mm Petri dish. Procedures forremoving paraffin from tissue sections are described in a variety ofspecialized handbooks well known to those skilled in the art.

To amplify a target nucleic acid sequence in a sample by PCR, thesequence must be accessible to the components of the amplificationsystem. One method of isolating target DNA is crude extraction which isuseful for relatively large samples. Briefly, mononuclear cells fromsamples of blood, amniocytes from amniotic fluid, cultured chorionicvillus cells, or the like are isolated by layering on a sterileFicoll-Hypaque gradient by standard procedures. Interphase cells arecollected and washed three times in sterile phosphate buffered salinebefore DNA extraction. If testing DNA from peripheral blood lymphocytes,an osmotic shock (treatment of the pellet for 10 sec with distilledwater) is suggested, followed by two additional washings if residual redblood cells are visible following the initial washes. This will preventthe inhibitory effect of the heme group carried by hemoglobin on the PCRreaction. If PCR testing is not performed immediately after samplecollection, aliquots of 10⁶ cells can be pelleted in sterile Eppendorftubes and the dry pellet frozen at −20° C. until use.

The cells are resuspended (10⁶ nucleated cells per 100 μl) in a bufferof 50 mM Tris-HCl (pH 8.3), 50 mM KCl 1.5 mM MgCl₂, 0.5% Tween 20, and0.5% NP40 supplemented with 100 μg/ml of proteinase K. After incubatingat 56° C. for 2 hr. the cells are heated to 95° C. for 10 min toinactivate the proteinase K and immediately moved to wet ice(snap-cool). If gross aggregates are present, another cycle of digestionin the same buffer should be undertaken. Ten μl of this extract is usedfor amplification.

When extracting DNA from tissues, e.g., chorionic villus cells orconfluent cultured cells, the amount of the above mentioned buffer withproteinase K may vary according to the size of the tissue sample. Theextract is incubated for 4-10 hrs at 50°-60° C. and then at 95° C. for10 minutes to inactivate the proteinase. During longer incubations,fresh proteinase K should be added after about 4 hr at the originalconcentration.

When the sample contains a small number of cells, extraction may beaccomplished by methods as described in Higuchi, “Simple and RapidPreparation of Samples for PCR”, in PCR Technology, Ehrlich, H. A.(ed.), Stockton Press, New York, which is incorporated herein byreference. PCR can be employed to amplify target regions in very smallnumbers of cells (1000-5000) derived from individual colonies from bonemarrow and peripheral blood cultures. The cells in the sample aresuspended in 20 μl of PCR lysis buffer (10 mM Tris-HCl (pH 8.3), 50 mMKCl, 2.5 mM MgCl₂, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20) andfrozen until use. When PCR is to be performed, 0.6 μl of proteinase K (2mg/ml) is added to the cells in the PCR lysis buffer. The sample is thenheated to about 60° C. and incubated for 1 hr. Digestion is stoppedthrough inactivation of the proteinase K by heating the samples to 95°C. for 10 min and then cooling on ice.

A relatively easy procedure for extracting DNA for PCR is a salting outprocedure adapted from the method described by Miller et al., NucleicAcids Res. 16:1215 (1988), which is incorporated herein by reference.Mononuclear cells are separated on a Ficoll-Hypaque gradient. The cellsare resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2mM Na₂ EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution of proteinase Kand 150 μl of a 20% SDS solution are added to the cells and thenincubated at 37° C. overnight. Rocking the tubes during incubation willimprove the digestion of the sample. If the proteinase K digestion isincomplete after overnight incubation (fragments are still visible), anadditional 50 μl of the 20 mg/ml proteinase K solution is mixed in thesolution and incubated for another night at 37° C. on a gently rockingor rotating platform. Following adequate digestion, one ml of a 6M NaClsolution is added to the sample and vigorously mixed. The resultingsolution is centrifuged for 15 minutes at 3000 rpm. The pellet containsthe precipitated cellular proteins, while the supernatant contains theDNA. The supernatant is removed to a 15 ml tube that contains 4 ml ofisopropanol. The contents of the tube are mixed gently until the waterand the alcohol phases have mixed and a white DNA precipitate hasformed. The DNA precipitate is removed and dipped in a solution of 70%ethanol and gently mixed. The DNA precipitate is removed from theethanol and air-dried. The precipitate is placed in distilled water anddissolved.

Kits for the extraction of high-molecular weight DNA for PCR include aGenomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis,Ind.), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md.),Elu-Quik DNA Purification Kit (Schleicher & Schuell, Keene, N.H.), DNAExtraction Kit (Stratagene, LaJolla, Calif.), TurboGen Isolation Kit(Invitrogen, San Diego, Calif.), and the like. Use of these kitsaccording to the manufacturer's instructions is generally acceptable forpurification of DNA prior to practicing the methods of the presentinvention.

The concentration and purity of the extracted DNA can be determined byspectrophotometric analysis of the absorbance of a diluted aliquot at260 nm and 280 nm. After extraction of the DNA, PCR amplification mayproceed. The first step of each cycle of the PCR involves the separationof the nucleic acid duplex formed by the primer extension. Once thestrands are separated, the next step in PCR involves hybridizing theseparated strands with primers that flank the target sequence. Theprimers are then extended to form complementary copies of the targetstrands. For successful PCR amplification, the primers are designed sothat the position at which each primer hybridizes along a duplexsequence is such that an extension product synthesized from one primer,when separated from the template (complement), serves as a template forthe extension of the other primer. The cycle of denaturation,hybridization, and extension is repeated as many times as necessary toobtain the desired amount of amplified nucleic acid.

In a particularly useful embodiment of PCR amplification, strandseparation is achieved by heating the reaction to a sufficiently hightemperature for a sufficient time to cause the denaturation of theduplex but not to cause an irreversible denaturation of the polymerase(see U.S. Pat. No. 4,965,188, incorporated herein by reference). Typicalheat denaturation involves temperatures ranging from about 80° C. to105° C. for times ranging from seconds to minutes. Strand separation,however, can be accomplished by any suitable denaturing method includingphysical, chemical, or enzymatic means. Strand separation may be inducedby a helicase, for example, or an enzyme capable of exhibiting helicaseactivity. For example, the enzyme RecA has helicase activity in thepresence of ATP. The reaction conditions suitable for strand separationby helicases are known in the art (see Kuhn Hoffman-Berling, 1978,CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev.Genetics 16:405-436, each of which is incorporated herein by reference).

Template-dependent extension of primers in PCR is catalyzed by apolymerizing agent in the presence of adequate amounts of fourdeoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP)in a reaction medium comprised of the appropriate salts, metal cations,and pH buffering systems. Suitable polymerizing agents are enzymes knownto catalyze template-dependent DNA synthesis. In some cases, the targetregions may encode at least a portion of a protein expressed by thecell. In this instance, mRNA may be used for amplification of the targetregion. Alternatively, PCR can be used to generate a cDNA library fromRNA for further amplification, the initial template for primer extensionis RNA. Polymerizing agents suitable for synthesizing a complementary,copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase(RT), such as avian myeloblastosis virus RT, Moloney murine leukemiavirus RT, or Thermus thermopliilus (Tth) DNA polymerase, a thermostableDNA polymerase with reverse transcriptase activity marketed by PerkinElmer Cetus, Inc. Typically, the genomic RNA template is heat degradedduring the first denaturation step after the initial reversetranscription step leaving only DNA template. Suitable polymerases foruse with a DNA template include, for example, E. coli DNA polymerase Ior its Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taqpolymerase, a heat-stable DNA polymerase isolated from Thermus aquaticusand commercially available from Perkin Elmer Cetus, Inc. The latterenzyme is widely used in the amplification and sequencing of nucleicacids. The reaction conditions for using Taq polymerase are known in theart and are described in Gelfand, 1989, PCR Technology, supra.

Allele Specific PCR

Allele-specific PCR differentiates between target regions differing inthe presence of absence of a variation or polymorphism. PCRamplification primers are chosen which bind only to certain alleles ofthe target sequence. This method is described by Gibbs, Nucleic AcidRes. 17:12427-2448 (1989).

Allele Specific Oligonucleotide Screening Methods

Further diagnostic screening methods employ the allele-specificoligonucleotide (ASO) screening methods, as described by Saiki et al.,Nature 324:163-166 (1986). Oligonucleotides with one or more base pairmismatches are generated for any particular allele. ASO screeningmethods detect mismatches between variant target genomic or PCRamplified DNA and non-mutant oligonucleotides, showing decreased bindingof the oligonucleotide relative to a mutant oligonucleotide.Oligonucleotide probes can be designed so that under low stringency,they will bind to both polymorphic forms of the allele, but at highstringency, bind to the allele to which they correspond. Alternatively,stringency conditions can be devised in which an essentially binaryresponse is obtained, i.e., an ASO corresponding to a variant form ofthe target gene will hybridize to that allele, and not to the wild-typeallele.

Ligase Mediated Allele Detection Method

Target regions of a test subject's DNA can be compared with targetregions in unaffected and affected family members by ligase-mediatedallele detection. See Landegren et al., Science 241:107-1080 (1988).Ligase may also be used to detect point mutations in the ligationamplification reaction described in Wu et al., Genomics 4:560-569(1989). The ligation amplification reaction (LAR) utilizes amplificationof specific DNA sequence using sequential rounds of template dependentligation as described in Wu, supra, and Barany, Proc. Nat. Acad. Sci.88:189-193 (1990).

Denaturing Gradient Gel Electrophoresis

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. DNA molecules melt in segments, termed melting domains,under conditions of increased temperature or denaturation. Each meltingdomain melts cooperatively at a distinct, base-specific meltingtemperature (T_(m)). Melting domains are at least 20 base pairs inlength, and may be up to several hundred base pairs in length.

Differentiation between alleles based on sequence specific meltingdomain differences can be assessed using polyacrylamide gelelectrophoresis, as described in Chapter 7 of Erlich, ed., PCRTechnology, “Principles and Applications for DNA Amplification”, W.H.Freeman and Co., New York (1992), the contents of which are herebyincorporated by reference.

Generally, a target region to be analyzed by denaturing gradient gelelectrophoresis is amplified using PCR primers flanking the targetregion. The amplified PCR product is applied to a polyacrylamide gelwith a linear denaturing gradient as described in Myers et al., Meth.Enzymol. 155:501-527 (1986), and Myers et al., in Genomic Analysis, APractical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95-139(1988), the contents of which are hereby incorporated by reference. Theelectrophoresis system is maintained at a temperature slightly below theTm of the melting domains of the target sequences.

In an alternative method of denaturing gradient gel electrophoresis, thetarget sequences may be initially attached to a stretch of GCnucleotides, termed a GC clamp, as described in Chapter 7 of Erlich,supra. Preferably, at least 80% of the nucleotides in the GC clamp areeither guanine or cytosine. Preferably, the GC clamp is at least 30bases long. This method is particularly suited to target sequences withhigh T_(m)'s.

Generally, the target region is amplified by the polymerase chainreaction as described above. One of the oligonucleotide PCR primerscarries at its 5′ end, the GC clamp region, at least 30 bases of the GCrich sequence, which is incorporated into the 5′ end of the targetregion during amplification. The resulting amplified target region isrun on an electrophoresis gel under denaturing gradient conditions asdescribed above. DNA fragments differing by a single base change willmigrate through the gel to different positions, which may be visualizedby ethidium bromide staining.

Temperature Gradient Gel Electrophoresis

Temperature gradient gel electrophoresis (TGGE) is based on the sameunderlying principles as denaturing gradient gel electrophoresis, exceptthe denaturing gradient is produced by differences in temperatureinstead of differences in the concentration of a chemical denaturant.Standard TGGE utilizes an electrophoresis apparatus with a temperaturegradient running along the electrophoresis path. As samples migratethrough a gel with a uniform concentration of a chemical denaturant,they encounter increasing temperatures. An alternative method of TGGE,temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses asteadily increasing temperature of the entire electrophoresis gel toachieve the same result. As the samples migrate through the gel thetemperature of the entire gel increases, leading the samples toencounter increasing temperature as they migrate through the gel.Preparation of samples, including PCR amplification with incorporationof a GC clamp, and visualization of products are the same as fordenaturing gradient gel electrophoresis.

Since-Strand Conformation Polymorphism Analysis.

Target sequences or alleles at the chosen boar taint loci can bedifferentiated using single-strand conformation polymorphism analysis,which identifies base differences by alteration in electrophoreticmigration of single-stranded PCR products, as described in Orita et al.,Proc. Nat. Acad. Sci. 85:2766-2770 (1989). Amplified PCR products can begenerated as described above, and heated or otherwise denatured, to formsingle-stranded amplification products. Single-stranded nucleic acidsmay refold or form secondary structures which are partially dependent onthe base sequence. Thus, electrophoretic mobility of single-strandedamplification products can detect base-sequence difference betweenalleles or target sequences.

Chemical or Enzymatic Cleavage of Mismatches

Differences between target sequences can also be detected bydifferential chemical cleavage of mismatched base pairs, as described inGrompe et al., Am. J. Hum. Genet. 48:212-222 (1991). In another method,differences between target sequences can be detected by enzymaticcleavage of mismatched base pairs, as described in Nelson et al., NatureGenetics 4:11-18 (1993). Briefly, genetic material from an animal and anaffected family member may be used to generate mismatch freeheterohybrid DNA duplexes. As used herein, “heterohybrid” means a DNAduplex strand comprising one strand of DNA from one animal, and a secondDNA strand from another animal, usually an animal differing in thephenotype for the trait of interest. Positive selection forheterohybrids free of mismatches allows determination of smallinsertions, deletions or other polymorphisms that may be associated withpolymorphisms.

Non-gel Systems

Other possible techniques include non-gel systems such as TAQMAN™(Perlin Elmer). In this system, oligonucleotide PCR primers are designedthat flank the mutation in question and allow PCR amplification of theregion. A third oligonucleotide probe is then designed to hybridize tothe region containing the base subject to change between differentalleles of the gene. This probe is labeled with fluorescent dyes at boththe 5′ and 3′ ends. These dyes are chosen such that while in thisproximity to each other the fluorescence of one of them is quenched bythe other and cannot be detected. Extension by Taq DNA polymerase fromthe PCR primer positioned 5′ on the template relative to the probe leadsto the cleavage of the dye attached to the 5′ end of the annealed probethrough the 5′ nuclease activity of the Taq DNA polymerase. This removesthe quenching effect allowing detection of the fluorescence from the dyeat the 3′ end of the probe. The discrimination between different DNAsequences arises through the fact that if the hybridization of the probeto the template molecule is not complete, i.e., there is a mismatch ofsome form, the cleavage of the dye does not take place. Thus, only ifthe nucleotide sequence of the oligonucleotide probe is completelycomplimentary to the template molecule to which it is bound willquenching be removed. A reaction mix can contain two different probesequences each designed against different alleles that might be presentthus allowing the detection of both alleles in one reaction.

Yet another technique includes an Invader Assay, which includesisothermic amplification that relies on a catalytic release offluorescence. See Third Wave Technology at www.twt.com.

Non-PCR Based DNA Diagnostics

The identification of a DNA sequence linked to sequences encodingenzymes involved in skatole metabolism can be made without anamplification step, based on polymorphisms including restrictionfragment length polymorphisms in an animal and a family member.Hybridization probes are generally oligonucleotides which bind throughcomplementary base pairing to all or part of a target nucleic acid.Probes typically bind target sequences lacking complete complementaritywith the probe sequence depending on the stringency of the hybridizationconditions. The probes are preferably labeled directly or indirectly,such that by assaying for the presence or absence of the probe, one candetect the presence or absence of the target sequence. Direct labelingmethods include radioisotope labeling, such as with P³² or S³⁵. Indirectlabeling methods include fluorescent tags, biotin complexes which may bebound to avidin or streptavidin, or peptide or protein tags. Visualdetection methods include photoluminescents, Texas red, rhodamine andits derivatives, red leuco dye and 3,3′,5,5′-tetramethylbenzidine (TMB),fluorescein, and its derivatives, dansyl, umbelliferone and the like orwith horse radish peroxidase, alkaline phosphatase and the like.

Hybridization probes include any nucleotide sequence capable ofhybridizing to the porcine chromosome where the CYP2A6 gene or othergene involved in skatole metabolism resides, and thus defining a geneticmarker linked to the gene, including a restriction fragment lengthpolymorphism, a hypervariable region, repetitive element, or a variablenumber tandem repeat. Hybridization probes can be any gene or a suitableanalog. Further suitable hybridization probes include exon fragments orportions of cDNAs or genes known to map to the relevant region of thechromosome.

Preferred tandem repeat hybridization probes for use according to thepresent invention are those that recognize a small number of fragmentsat a specific locus at high stringency hybridization conditions, or thatrecognize a larger number of fragments at that locus when the stringencyconditions are lowered.

One or more additional restriction enzymes and/or probes and/or primerscan be used. Additional enzymes, constructed probes, and primers can bedetermined by routine experimentation by those of ordinary skill in theart and are intended to be within the scope of the invention.

According to the invention, polymorphisms in genes encoding enzymesinvolved in skatole metabolism have been identified which have anassociation with boar taint. The presence or absence of the markers, inone embodiment may be assayed by PCR-RFLP analysis using the restrictionendonucleases and amplification primers may be designed using analogoushuman, pig or other sequences due to the high homology in the regionsurrounding the polymorphisms, or may be designed using known genesequence data as exemplified in GenBank or even designed from sequencesobtained from linkage data from closely surrounding genes based upon theteachings and references herein. The sequences surrounding thepolymorphism will facilitate the development of alternate PCR tests inwhich a primer of about 4-30 contiguous bases taken from the sequenceimmediately adjacent to the polymorphism is used in connection with apolymerase chain reaction to greatly amplify the region before treatmentwith the desired restriction enzyme. The primers need not be the exactcomplement; substantially equivalent sequences are acceptable. Thedesign of primers for amplification by PCR is known to those of skill inthe art and is discussed in detail in Ausubel (ed.), Short Protocols inMolecular Biology, 4th Edition, John Wiley and Sons (1999).

The following is a brief description of primer design.

Primer Design Strategy

Increased use of polymerase chain reaction (PCR) methods has stimulatedthe development of many programs to aid in the design or selection ofoligonucleotides used as primers for PCR. Four examples of such programsthat are freely available via the Internet are: PRIMER by Mark Daly andSteve Lincoln of the Whitehead Institute (UNIX, VMS, DOS, andMacintosh), Oligonucleotide Selection Program (OSP) by Phil Green andLaDeana Hiller of Washington University in St. Louis (UNIX, VMS, DOS,and Macintosh), PGEN by Yoshi (DOS only), and Amplify by Bill Engels ofthe University of Wisconsin (Macintosh only). Generally these programshelp in the design of PCR primers by searching for bits of knownrepeated-sequence elements and then optimizing the T_(m) by analyzingthe length and GC content of a putative primer. Commercial software isalso available and primer selection procedures are rapidly beingincluded in most general sequence analysis packages.

Sequencing and PCR Primers

Designing oligonucleotides for use as either sequencing or PCR primersrequires selection of an appropriate sequence that specificallyrecognizes the target, and then testing the sequence to eliminate thepossibility that the oligonucleotide will have a stable secondarystructure. Inverted repeats in the sequence can be identified using arepeat-identification or RNA-folding program such as those describedabove. If a possible stem structure is observed, the sequence of theprimer can be shifted a few nucleotides in either direction to minimizethe predicted secondary structure. The sequence of the oligonucleotideshould also be compared with the sequences of both strands of theappropriate vector and insert DNA. Obviously, a sequencing primer shouldonly have a single match to the target DNA. It is also advisable toexclude primers that have only a single mismatch with an undesiredtarget DNA sequence. For PCR primers used to amplify genomic DNA, theprimer sequence should be compared to the sequences in the GenBankdatabase to determine if any significant matches occur. If theoligonucleotide sequence is present in any known DNA sequence or, moreimportantly, in any known repetitive elements, the primer sequenceshould be changed.

The methods and materials of the invention may also be used moregenerally to evaluate pig DNA, genetically type individual pigs, anddetect genetic differences in pigs. In particular, a sample of piggenomic DNA may be evaluated by reference to one or more controls todetermine if a polymorphism in the particular gene is present.Preferably, RFLP analysis is performed with respect to the pig gene, andthe results are compared with a control. The control is the result of aRFLP analysis of the pig gene of a different pig where thepolymorphism(s) of the pig gene is/are known. Similarly, the genotype ofa pig may be determined by obtaining a sample of its genomic DNA,conducting RFLP analysis of the gene in the DNA, and comparing theresults with a control. Again, the control is the result of RFLPanalysis of the gene of a different pig. The results genetically typethe pig by specifying the polymorphism(s) in its genes. Finally, geneticdifferences among pigs can be detected by obtaining samples of thegenomic DNA from at least two pigs, identifying the presence or absenceof a polymorphism in the gene, and comparing the results.

These assays are useful for identifying the genetic markers relating toboar taint, as discussed above, for identifying other polymorphisms inthe genes encoding enzymes involved in skatole metabolism and for thegeneral scientific analysis of pig genotypes and phenotypes.

The examples and methods herein disclose certain gene(s) which has beenidentified to have a polymorphism(s) which is associated eitherpositively or negatively with a beneficial trait that will have aneffect on meat quality, heavy muscling, and/or skeletal muscle crampingdisease for animals carrying this polymorphism. The identification ofthe existence of a polymorphism within a gene is often made by a singlebase alternative that results in a restriction site in certain allelicforms. A certain allele, however, as demonstrated and discussed herein,may have a number of base changes associated with it that could beassayed for which are indicative of the same polymorphism (allele).Further, other genetic markers or genes may be linked to thepolymorphisms disclosed herein so that assays may involve identificationof other genes or gene fragments, but which ultimately rely upon geneticcharacterization of animals for the same polymorphism. Any assay whichsorts and identifies animals based upon the allelic differencesdisclosed herein are intended to be included within the scope of thisinvention. One of skill in the art, once a polymorphism has beenidentified and a correlation to a particular trait established willunderstand that there are many ways to genotype animals for thispolymorphism. The design of such alternative tests merely representsoptimization of parameters known to those of skill in the art and isintended to be within the scope of this invention as fully describedherein.

Example 5

Cloning, Expression and Functional Characterization of Cytochrome P4502A6 Gene from Pig Liver

Entire male pigs are used for meat production in pig industry, due to abetter feed conversion, improved carcass leanness and addressed animalwelfare. Therefore, raising male pigs may improve the profitability ofpork production by up to 30% (Babol et al. 1995). However, the frequentoccurrence of off-odors in cooked pork from uncastrated male pigs,commonly known as “boar taint”, is highly objectionable to consumers.Skatole is one of the major contributors to boar taint (Gonzalo et al.2000). Skatole is absorbed from the gut and then metabolized primarilyin the liver. In pigs, cytochrome P450 enzymes have been found to havesignificant impact on metabolism of skatole. It has been shown thatCYP2A6 is one of major key enzymes in the metabolism of skatole (Gonzaloet al. 2000). In pigs, CYP2A6 has been found to be highly and negativelycorrelated with skatole accumulation in fat (Babol et al. 1998; Gonzaloet al. 2000). Therefore CYP2A6 plays an important role in the metabolismand clearance of skatole from the body in pigs.

Cytochrome P450 is a superfamily of hemoprotein (Ingelman-Sundberg et al1999). In human, CYP2A6 is predominantly expressed in the liver (Koskelaet al. 1999; Oscarson, 2001). It is a major hepatic member of thefamily, which metabolizes pharmaceutical (Miles et al. 1990) and manyother drugs and environment chemicals (Yamazaki et al 1992). In human,CYP2A6 was first identified as the coumarin-7 hydroxylase (Yamano etal., 1990), and has received a lot of attention since then, due to itsprinciple role in nicotine C-oxidation and possible involvement insmoking behavior and lung cancer susceptibility (Xu et al., 2002;Oscarson, 2001). The knowledge concerning CYP2A6 in human hassubstantially increased. However, the information about the CYP2A6 gene,its expression and how a genetic variant of CYP2A6 affect skatole levelin pigs is remains empty.

In present study, we constructed the cDNA library from pig liver byrapid amplification of cDNA ends (RACE) method and reported the sequenceof porcine CYP2A6 cDNA. We examined the expression pattern of the CYP2A6mRNA species in different tissues in pigs by Northern analysis.Polymerase chain reaction technique combined with single strandconformational polymorphism (PCR-SSCP) was used to scan and identify anygenetic polymorphism of CYP2A6 coding region from porcine liver tissues,which may alter the metabolic capacities of the enzyme. Furthermore,functional studies with this genetic polymorphism of CYP2A6 were carriedout.

Tissue samples

Liver tissues were obtained from a male pig for construction of cDNAlibrary. To identify any genetic polymorphism in CYP2A6, sixty-nine pigsfrom a variety of breeds, including Yorkshire, Duroc, Landrace, andPietrain, as well as crosses between Landrace and Duroc, Large White andDuroc, and Large White and Pertain, were slaughtered at an average liveweight of 144 kg (144 kg±33) at the Department of Animal and PoultryScience abattoir. A sample of liver was taken immediately followingexsanguinations, frozen in liquid nitrogen and stored at −70 C for untiluse.

Isolation of Total RNA

One hundred milligram of tissue samples were homogenized in 1 ml ofTri-Reagent (Sigma) and incubated for 10 min at room temperature. Afterincubation, 0.2 ml of chloroform was added and vortexed. The sampleswere centrifuged at 12,000×g for 10 min at 4° C. and then aqueous phasewas transferred in to a sterile tube. The aqueous phase was mixed with0.5 ml of isopropanol and incubated at room temperature for 10 min toprecipitate the RNA. Pellet was obtained by centrifugation (12,000×g for10 min at 4° C.). The pellet was washed with 75% ethanol and thensuspended into 50 μl of DEPC water.

Construction and Screening of a pig cDNA RACE Library

5′ and 3′ rapid amplification of cDNAs (RACE) were constructed from 1 μgof total RNA from liver separately by use of Smart RACE cDNAAmplification kit (Clontech), and used as templates in the subsequentPCR screening of porcine CYP2A6. The 5′RACE was performed bysynthesizing the first strand cDNA with a modified lock-docking oligo(dT) primer and then tailing the product 5′AAG CAG TGG TAT CAA CGC AGAGTA CGC GGG 3′(SEQ ID NO:9) (anchor primer) in 5′end via terminaltransferase. The 3′ RACE was performed with oligo (dT) primer butincludes the same lock-docking nucleotide positions as in 5′RACE. Thefirst fragment of CYP2A6 was amplified with the primers designed fromthe conserved region of human 2A6, mouse 2A5, and rat 2A3 cDNA sequence.The forward primer is 5′ AGG ACA AAG AGT TCC TGT CAC TG 3′, (SEQ IDNO:10) reverse primer is 5° CAA TCT CCT CAT GGA CCT TGG 3′(SEQ IDNO:11). To obtain full-length porcine CYP2A6, following primers wereused in the subsequent PCR-based screening: 5′ ATG AGC AGC AGG AAG CCGTAG 3′(SEQ ID NO:12) and anchor primer with 5′Race as a template; 5′ CTACGG CTT CCT GCT GCT CAT 3′(SEQ ID NO:13) and anchor primer with 3′Raceas a template; 5′CAC AAC GAT GCG CTA CGG CT 3′(SEQ ID NO:14) and 5′GCAGGAAGCTCATGGTGTAG 3′(SEQ ID NO:15) with either 3′ or 5′Race as atemplate. The PCR consisted of 35 cycles of denaturing for 1 minute at94° C., optimal annealing for I minute, and extending for 1 minute, witha final 10 minutes extension step at 72° C. 10 μl of the PCR productswere analyzed by electrophoresis on a 1% agarose gel.

Colony Hybridization

When there were multiple bands to be amplified from both 3′and 5′Racetemplates, the PCR products were cloned into pGEM-T Easy Vector System(Promega), and then subjected to colonies hybridization to confirm thespecificity of amplified fragment prior to DNA sequencing. Colonies werelifted up to positively charged nylon membrane (Roche), then subjectedto lysis and fixation in 0.5M NaCl for 5 minutes, rinsing in 5×SSC for 1minutes, and air dry; Colonies hybridization was performed with ECLnucleotide DNA labeling and detection kit (Amersham Life Science). Theprobe used in the hybridization was the fragment first amplified by theprimers designed from the human 2A6, mouse 2A5, and rat 2A3 cDNAconserved region. After hybridization overnight at 42° C., the membranewas washed with 0.15×SSC for 20 minutes twice and exposed to x-ray film(Kodak). The colony that gives the strongest signal is subjected to besequencing.

Isolation of Full-Length Porcine CYP2A6 cDNA

To obtain fill-length porcine CYP2A6 sequence, forward primer 5′ CTC GCAGTG CCA CCA TGC TG 3′ (SEQ ID NO:16) and reverse primer 5′ GCA GGA AGCTCA TGG TGT AGG TC (SEQ ID NO: 17) 3′ were designed based on thesequence obtained from the 5′ and 3′ RACE, and used to amplify thefull-length porcine CYP 2A6 either with 5′ or 3′ RACE cDNA as atemplate. PCR profile was 3 min at 94° C., followed by 35 cycles of 1min at 94° C., 1 min 30 sec at 64° C., 2 min at 72° C. and fin 10 min at72° C. and two drops of mineral oil were added. The PCR fragment wascloned into T-Easy vector (Promega) and subjected to sequence analysis.

Northern Blot Analysis

Total RNAs were isolated from porcine spleen, thymus, liver, lung,muscle, ovary, kidney, small intestine, heart, and testis tissues withTri-Reagent (Sigma). 20 μg of total RNA from each tissue was subjectedto electrophoresis in the 2.1M formaldehyde-containing 1% agarose geland transferred to nylon membrane (Amersham Pharmacia Biotech) withdownward capillary. Full-length of the porcine CYP2A6 (1498bp) wascreated from forward primer 5′ CTC GCA GTG CCA CCA TGC TG 3′(SEQ IDNO:16) and reverse primer5′ GCA GGA AGC TCA TGG TGT AGG TC 3′(SEQ IDNO:17) from pig liver cDNA library we created. CYP2A6 cDNA was labeledusing random primers with digoxigenin-dUTP (Roche MolecularBiochemicals) and hybridized at 50° C. overnight. After prewashing with2×SSC containing 0.1% SDS, the membrane was washed with 0.2×SSCcontaining 0.1% SDS for 15 minutes twice at 67°. The hybridized probesare immunodetected with anti-digoxigenin-alkaline phosphatase conjugate,detected with the colorimetric substrates (DIG, Roche), and exposed toKodak Scientific Imaging film (Kodak) for 1 hour at room temperature.

Sequencing Analysis

The PCR fragments were ligated into pGEM-T Easy Vector System (Promega),and then transformed into competent DH5α cells. DNAs were purified andsubject to sequencing using an Applied Biosystems model ABI 377 DNAsequencer.

RT-PCR

To scan any genetic polymorphism in the CYP2A6 from individuals, RT-PCRproducts that cover its whole coding region were amplified and thensubjected to SSCP analysis. First strand cDNA was synthesized from 1 to5 μg of total RNA from liver samples using SuperScript reversetranscriptase (Invitrogen) and oligo (dT) primer (Sigma). Following thereverse transcription, 2.5 μl of the first strand cDNA was used as thetemplate for PCR. The PCR mixtures (50 ul) contained 1× PCR buffer (100mM Tris-HCl, pH 8.3; 500 mM KCl, 11 mM MgCl₂, 0.1% gelatin), 0.2 mMdNTP, 0.4 mM primers (forward and reverse primer) and 2.5 U of Red Taqpolymerase (Sigma). The primer pair (forward primer, 5′ CTC GCA GTG CCACCA TGC TG 3′, (SEQ ID NO:16) reverse sequence, 5′ GCA GGA AGC TCA TGGTGT AGG TC 3′) (SEQ ID NO:17) was designed to amplify the entire codingregion of porcine CYP2A6, based on our isolated CYP2A6 (GenBankaccession number AY091516). The PCR profile was 3 min at 94° C.,followed by 35 cycles of 1 min at 94° C., 1 min at 65° C., 1 min at 72°C. and final extension of 10 min at 72° C.

Single-Strand Conformational Polymorphism Analysis

PCR products were first cut into fragments with BstxI enzyme, and thenresolved by SSCP analysis. 5 μl of PCR product amplified was digestedwith BstxI in 20 μl reaction at 37° C. for 3 hours. A total of 7 μl ofdigested fragments were then diluted with 13 μl of loading buffer (10%of Sucrose, 0.01% of Bromophenol blue and 0.01% of Xylene cyanol FF).Each digestion reaction was denatured at 100° C. for 5 min, chilled onice and resolved on 10% of polyacrylarnide gel. The electrophoresis wascarried in a vertical unit (Bio-Rad Laboratories, 130×160×1.0 mm), in0.6× TBE buffer for 17 hours at 15° C. at 160 V. The gels were thensilver stained.

CYP2A6 Activity

CYP2A6 activity is assayed by measurement of coumarin 7-hydroxylaseactivity on pig liver microsomal samples. 20 μl of microsomal suspensioncontaining 0.4 mg microsomal protein were mixed with 200 μl of coumarinhydroxylase reaction mix (0.05M Tris buffer pH 7.4, 5mM MgCl₂ and 0.2 mMcoumarin). The reaction was started by adding 15 μl of 25 mM NADPH.After incubation at 37° C. for 15 minutes, the reaction was stopped bythe addition of 50 μl of 20% trichloroacetic acid, followed bycenrifugation at 10,000 g for 2 min. Two hundred microliters of thesupernatant fraction was diluted with 2 ml of 0.1 M Tris buffer (pH9.0), and the fluorescence-was determined at wavelengths of 390 nm forexcitation at and 440nm for emission.

Measurement of Skatole Level in Fat

A backfat sample was collected at the midline point of 11th rib andfrozen at −20° C. until assayed for skatole. The skatole content wasmeasured with a colorimetric assay, according to the method described byGonzalo et al. (2000).

Western Analysis

Liver tissue (1 g) was homogenized in 5 ml of sample buffer (1% cholicacid, 0.1% SDS in PBS buffer) and the protein concentrations ofhomogenates were determined using the BCA kit (Pierce). 40 μg of totalprotein were subjected to sodium dodecyl sulphate gel electrophoresisusing a 12% polyacrylamide gel. The protein was transferred to anitro-cellulose filter (BioRad), incubated with mouse anti-humanmonoclonal 2A6-antibody MAB-2A6 (Gentest), and subsequently anti-mouseIGG peroxidase conjugate developed in goat (Sigma). Immunoreactive bandswere stained by a chemiluminescence procedure (ECL, Amersham LifeScience) and visualized by autoradiography.

The CYP2A6 cDNA Sequence and Sequence Characterization

Pig CYP2A6 cDNA was isolated by PCR screening of the liver cDNA libraryconstructed with RACE. The nucleotide sequence of the CYP2A6 cDNA was1519 bp long and contained a 1485 bp-long open reading frame (ORF),which encodes 497 amino acids (FIG. 12). Pig CYP2A6 cDNA sequence wassubmitted to Genbank database under the accession number AY091516.

The human CYP2A6, mouse CYP2A5, rat CYP2A3 were identified as thecoumarin-7 hydroxylase. We compared pig CYP2A6 ORF to above genes, itshowed 87% homology to human CYP2A6, 85% to mouse CYP2A5, and 86% to rat2A3. The deduced amino acid sequence for pig CYP2A6 showed 87% homologyto human 2A6, 90% to mouse 2A5, and 89% to rat 2A3 (FIG. 13). In humanCYP2A6, Gln104, Phe209 and His477 were reported to be active sites forCYP2A6 coumarin 7-hydroxylase activity, oxidative metabolism of nicotineand cotinine (Lewis et al. 1999). R128 was represents one of key bindingresidues for human CYP2A6 (Kiragawa et al, 2001; Lewis et al, 1999). Allabove active sites are conserved in the putative pig CYP2A6.

Expression of CYP2A6 mRNA Species in Various Tissues

The expression patterns of CYP2A6 mRNA in various tissues, includingspleen, thymus, liver, lung, muscle, ovary, kidney, small intestineheart and testis from pigs, were investigated by Northern blotting byusing pig CYP2A6 cDNA as a probe. The result showed that CYP2A6 are onlyexpressed in liver and kidney tissue (FIG. 14). A much higher level ofCYP2A6 mRNA was observed in the liver, and a lower level of CYP2A6 mRNAwas expressed in the kidney. The result showed the CYP2A6 ispredominantly expressed in pig liver tissue. It indicated the liver isthe major tissue that plays important role in CYP2A6 metabolism in pigs.

CYP2A6 Genetic Polymorphism

In order to identify any genetic polymorphism of CYP2A6, which may alterthe metabolic capacities of the enzyme, polymerase chain reactiontechnique combined with single strand conformational polymorphism(PCR-SSCP) was used to scan CYP2A6 coding region from porcine livertissues. In pig, CYP2A6 full-length cDNA was amplified by PCR withprimer pair: forward primer 5′ CTC GCA GTG CCA CCA TGC TG 3′ (SEQ IDNO:16) and reverse primer 5′ GCA GGA AGC TCA TGG TGT AGG TC 3′ (SEQ IDNO:17) from liver tissues. The resulting PCR products were about 1500 bpin size. Digested PCR products with BstxI were subjected to SSCPanalysis using our optimized system. We found that there are severaldifferent polymorphisms existing in CYP2A6 coding region (data notshown). Of which, one of deletion that resulted in coding region frameshifting received our most attention. Due to one G missing, the lengthof ORF region of CYP2A6 changes from 1485 bp to 612 bp. This also causesthe length of its encoded gene product change from 495 amino acid to 204amino acid. It is suggested that the deletion might also result ininactivation of CYP2A6 activity for the individual that contains suchdeletion. It has been shown that CYP2A6 is one of major key enzymes inthe metabolism of skatole (Gonzalo et al. 2000). CYP2A6 is negativelycorrelated with skatole accumulation in fat (Babol et al. 1998).Therefore, we infer that CYP2A6 activity for the sample that exists suchdeletion would be zero for its comarin 7-hydroxylase activity due tocoding region frame shifting of CYP2A6 gene, and that skatole levelshould be higher due to losing this enzyme activity to clear skatolefrom the body.

To evaluate above hypothesis and investigate the association of thisgenetic polymorphism of CYP2A6 with skatole level, the phenotyping usingskatole level measurement, coumarin 7-hydroxylase activity assay andimmunoblotted with monoclonal anti-human CYP2A6 anti-body (Gentest) forthe samples showed different genotype, were further carried out. Theresults showed that the skatole level is much higher for the sample withdeleted mutation than that in wild type samples. Coumarin 7-hydroxylaseassay and immunoblotting analysis also told us zero for coumarin7-hydroxylase activity and negative immunoreactive band for the samplethat has deleted mutation, while remaining lower skatole level, higheractivity and detectable immunoreactive bands for wild type samples (FIG.15). The results are strongly supporting our suggestion that the CYP2A6deletion caused a complete lack of enzymatic activity, and hence causedhigher level skatole level in pig.

In human, CPY2A6 gene has been extensively studied; however, theinformation about the CYP2A6 gene, its expression and how a geneticvariant of CYP2A6 affect skatole level in pigs is remains empty. In thisstudy, we reported the molecular cloning, functional characterization ofCYP2A6 gene in pig. We designed the primers based on conserved region ofhuman 2A6, mouse 2A5 and rat 2A3 cDNA sequence. Coumarin 7-hydroxylationis catalysed by a high-affinity CYP2A6 and CYP2A5 enzyme in human andmouse (Miles et al., 1990; Donato et al., 2000), and that by CYP2A3 inrat. The formation of 7-hydroxycoumarin has been used as an in vivo andinvitro probe for CYP2A6 in human, CYP2A5 in mouse, and 2A3 in rat(Rodrigues et al., 1994; Rautio et al., 1992; Fernandez-Salguero et al,1995). Therefore, by using the designed primers, we screened out thefirst fragment, subsequently the whole sequence of pig CYP2A6 cDNA.

The CYP2A6 in human, CYP2A5 in mouse, and CYP2A3 was sequenced (Accessnumber: U22027 for human, BC046605 for mouse, and M33190 for rat), andhas been mapped to chromosome 19q13.2 (b: Fernandez-Salguero et al.,1995) chromosome 7 (Kent et al.,1987) and chromosome 1 (STS: D1Mgh28),respectively. As indicated in the results, when comparing pig CYP 2A6sequence to its orthologous genes, sequence of human CYP2A6, mouse 2A5and rat CYP2A3, we found that it has high homology to its orthologs bothin cDNA sequence and amino acid sequence. And all the important activesites of amino acid sequence in human CYP.2A6 are also conserved in ourputative pig CYP2A6 sequence. Furthermore, we searched against human,mouse and rat genomic database with pig CYP2A6 cDNA sequence, we foundthat pig CYP2A6 only hit a human genomic clone NT_(—)011109) fromchromosome 19q13.2, mouse genonlic clone (NT_(—)039410) from chromosome7, and rat genomic clone (NW_(—)043361) from chromosome 1q21,respectively. The hit scores showed that pig CYP2A6 cDNA sequence hashighest identity with human CYP2A6 genomic clone (91%) at humanchromosome 19q13.2, with mouse 2A5 genomic clone (89%) at mousechromosome 7, and with rat CYP2A3 genomic clone (88%) at rat chromosome1q21. All these findings taken together thus led us to conclude that theputative CYP2A6 is indeed pig CYP2A6.

In this study, we performed northern blot analysis for pig CYP2A6 mRNAdistribution in different tissues, the results showed that CYP2A6 isexpressed predominantly in liver and at a much higher level in liver,lower level in kidney. This indicated liver is the most important tissuefor clearance of skatole from body in pig. In spite of high similaritiesof pig CYP2A6 with its orthologous genes, these enzymes differ in tissuedistribution. It has been reported that mRNA expression is observedmainly in liver for human 2A6 (Koskela et al. 1999; Oscarson, 2001), inliver, kidney and small intestine for mouse 2A5 (Su et al., 1998), andin olfactory mucosa and lung for rat 2A3 (Su, et al., 1996; Kimura etal., 1989). In our study, we found that CYP2A6 is not expressed in smallintestine and lung in pig. The difference of expression for CYP2A6 mRNAand its orthologous genes in various tissues suggest there might bedifference in their promoter region, this difference may be useful forstudy regulation of tissue-specific gene expression.

In human, CYP2A6 has been one of most important enzyme in nicotineC-oxisation, due to the important of CYP2A6 in nicotine metabolism, andpossible involvement in smoking behavior and lung cancer susceptibilityQ(u et al., 2002; Oscarson, 2001). Polymorphism in the human CYP2A6 genemay thus impact on both smoking behavior and lung cancer susceptibility.Therefore, substantial efforts have been focused on detecting geneticpolymorphism and its consequences (Paschke, et al., 2001; Kamataki, etal., 1999; Oscarson, et al., 2001; Kitagawa, et al., 2001). In human,large interindividual differences has been seen in the levels of CYP2A6enzyme, due to the genetic variants mainly located in the open readingframe (Nakajima et al., 2002). A number of genetic polymorphisms havebeen detected for the CYP2A6 in human, including SNPs in the codingregion that lead to inactivation, such as Gly479Val (Oscarson et al.,2001) and Arg128Gln (Kitagawa et al., 2001). The progress in suchresearches will facilitate molecular study to clarify how critical theCYP2A6 polymorphism in causing genetic difference and its subsequentconsequence.

The role of cytochrome P450 enzyme including CYP2A6 in the metabolism ofskatole has been investigated in human, mouse, and rabbit(Thornton-Manning et al., 1996). In pigs, It has been reported thatCYP2A6 is one of key enzymes in the hepatic metabolism of skatole(Gonzalo et al. 2000) and CYP2A6 is negatively correlated with skatoleaccumulation in fat (Babol et al. 1998). Therefore, pigs with highlevels of these enzyme incuding CYP2A6 have low levels of skatole in thefat, since skatole is rapidly metabolised and celared from the body,pigs with low levels of these enzyme can have high levels of skatole inthe fat. Therefore, CYP2A6 could be use as an genetic marker to selectagainst skatole, once CYP2A6 genetic variant and its consequence onskatole has been investigated. Because there is no information on CYP2A6gene, we first isolated pig CYP2A6 from liver tissue using RACE method,then performed PCR-SSCP analysis to scan pig CYP2A6 coding region basedon our optimized genotyping system. In this study, we focus our effortson evaluation of CYP2A6 functional region and its genetic polymorphism.We have identified one genetic polymorphism, resulting in a frameshifting in the coding region and inactivation of the enzyme activity.Due to deletion of CYP2A6, coumarin 7-hydroxylation and CYP2A6 geneproduct are not detectable. It is not known at which age theunregulation of CYP2A6 occurs. In our CYP2A6 phenotyping studies usingcoumarin, western analysis with mouse anti-human monoclonal2A6-antibody, and skatole measurement in pig, we also found that thereare the existence of additional alleles outside of coding region thatmodulate or inactive CYP2A6 activity (data not shown). Therefore, itwould be helpful to investigate the promoter region of CYP2A6, incombination with phenotype individuals with either coumarin,immunodectected band as indicators of in vivo and invitro CYP2A6activity in future study, since there may be other CYP2A6 alleles thathave not yet known.

In this study, we isolated pig CYP2A6 cDNA from liver and found theCYP2A6 deletion in ORF region, which resulted in a complete lack of theenzymatic activity. There has been no published study that investigatesthe impact of genetic polymorphism in CYP2A6 on its clearance of skatolefrom body in pig. The data presented in this study suggest that theCYP2A6 gene deletion might play an important role in the development ofgenetic marker for skatole.

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While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A method of genetically typing animals to determine those withdesired boar taint characteristics, comprising: obtaining a sample ofgenetic material from said animal; and assaying for the presence of agenotype in said animal which is associated with improved boar taint,said genotype characterized by the following: a) a polymorphism in theCYP2A6 gene, said polymorphism being one which is associated withimproved boar taint characteristics.
 2. The method of claim 1 whereinsaid polymorphism is a t/c polymorphism at nucleotide position 124 ofSEQ ID NO:3.
 3. The method of claim 1 wherein-said polymorphism is adeletion of guanine at nucleotide position 422 of SEQ ID NO:3.
 4. Themethod of claim 1 wherein said polymorphism results in a loss offunction mutation of CYP2A6.
 5. The method of claim 1 wherein saidpolymorphism results in a Phe to Leu change at position 42 of SEQ IDNO:4.
 6. The method of claim 1 wherein said polymorphism results in atruncated CYP2A6 protein of SEQ ID NO:8.
 7. The method of claim 1wherein said step of assaying is selected from the group consisting of:restriction fragment length polymorphism (RFLP) analysis,minisequencing, MALD-TOF, SINE, heteroduplex analysis, one baseextension methods, single strand conformational polymorphism (SSCP),denaturing gradient gel electrophoresis (DGGE) and temperature gradientgel electrophoresis (TGGE).
 8. A method of genetically typing animalsaccording to skatole metabolism comprising: obtaining a sample ofgenetic material from said animal; assaying for the presence of anallele characterized by a polymorphism in a CYP2A6 gene present in saidsample, and correlating said allele with skatole metabolism andconcomitant boar taint.
 9. The method of claim 8 wherein saidpolymorphism results in a deletion of guanine at position 422 of SEQ IDNO:3, or a c/t transition at position 124 of SEQ ID NO:3.
 10. The methodof claim 8 wherein said step of assaying is selected from the groupconsisting of: restriction fragment length polymorphism (RFLP) analysis,minisequencing, MALD-TOF, SINE, heteroduplex analysis, one baseextension methods, single strand conformational polymorphism (SSCP),denaturing gradient gel electrophoresis (DGGE) and temperature gradientgel electrophoresis (TGGE).
 11. The method of claim 9 further comprisingthe step of amplifying the amount of CYP2A1 gene or a portion thereofwhich contains said polymorphism.
 12. A method of determining geneticvariability in animals which is linked to skatole metabolism comprising:obtaining a biological sample from a group, line, population or familyof animals, said sample comprising a nucleotide sequence encoding anenzyme associated with cytochrome P450 metabolism; comparing saidsequence to a reference sequence to identify a polymorphism; correlatingsaid polymorphism with variability in skatole metabolism.
 13. A methodof screening animals to determine those with desired boar taintcharacteristics, comprising: obtaining a sample of genetic material fromsaid animal; and assaying for the presence of a genotype in said animalwhich is associated with improved boar taint, said genotypecharacterized by the following: a) a polymorphism in a cytochrome CYP450gene, said polymorphism being one which is associated with improved boartaint characteristics.
 14. A nucleotide sequence which encodes atruncated CYP2A6 protein, having an deletion of the guanine at position422 of SEQ ID NO:3 or its equivalent as determined by BLAST, saidnucleotide sequence comprising one or more of the following: (a) SEQ IDNO: 3, or SEQ ID NO:7, (b) a sequence which will hybridize underconditions of high stringency to the sequences in (a); or (c) a sequencewith at least about 90% sequence identity to the sequences in (a).
 15. Atruncated CYP2A6 protein according to claim
 14. 16. A nucleotidesequence which encodes a CYP2A6 protein, having an LEU 42 PHE mutationof SEQ ID NO:3 or its equivalent as determined by BLAST said nucleotidesequence comprising one of the following: (a) SEQ ID NO: 1 or SEQ IDNO:5, (b) a sequence which will hybridize under conditions of highstringency to the sequences in (a); or (c) a sequence with at leastabout 90% sequence identity to the sequences in (a).
 17. A CYP2A6skeletal muscle protein, said protein comprising an amino acid sequencecomprising one of the following: (a) SEQ ID NO: 2, 4, 6, or 8 (b)conservatively modified variant of (a), or (c) a sequence with at leastabout 80% homology to a sequence in (a)
 18. A nucleotide sequenceencoding the protein of claim
 17. 19. A porcine CYP2A6 protein,comprising the following: a) SEQ ID NO:4 b) conservatively modifiedvariants of SEQ ID NO:4 c) a sequence with 80% homology to SEQ ID NO:420. A nucleotide sequence encoding a CYP2A6 protein comprising: a) asequence encoding a protein of claim 19 b) SEQ ID NO: 3 c) a sequencewith 90% sequence identity to SEQ ID NO:3 d) a sequence which willhybridize under conditions of high stringency to the complement of SEQID NO:3 e) the complement of any of a-d.